Single chain variable fragment cd3 binding proteins

ABSTRACT

Disclosed herein are single chain variable fragment CD3 binding proteins with improved binding affinities, and robust aggregation profiles. Also described are multispecific binding proteins comprising a single chain variable fragment CD3 binding protein according to the instant disclosure. Pharmaceutical compositions comprising the binding proteins disclosed herein and methods of using such formulations are provided.

CROSS-REFERENCE

This is a continuation application of U.S. application Ser. No.15/600,264, filed May 19, 2017, and claims the benefit of U.S.Provisional Application No. 62/339,685 filed May 20, 2016, all of whichare incorporated by reference herein in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 19, 2017, isnamed 47517-704_201_SL.TXT and is 66,042 bytes in size.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference,and as if set forth in their entireties.

BACKGROUND OF THE INVENTION

CD3 is a homodimeric or heterodimeric antigen expressed on T cells inassociation with the T cell receptor complex (TCR) and is required for Tcell activation. Anti-CD3 antibodies have therapeutic purposes involvingthe activation of T cells. Present disclosure provides single chainvariable fragment CD3 binding proteins, including multispecificantibodies containing the same.

SUMMARY OF THE INVENTION

In one embodiment is disclosed a single chain variable fragment CD3binding protein, comprising a variable heavy chain region (VH), avariable light chain region (VL), and a linker, wherein VH comprisescomplementarity determining regions HC CDR1, HC CDR2, and HC CDR3,wherein VL comprises complementarity determining regions LC CDR1, LCCDR2, and LC CDR3, wherein (a) the amino acid sequence of HC CDR1 is asset forth in GX₁X₂X₃NX₄YX₅X₆N (SEQ ID NO. 2), X₁ is phenylalanine orasparagine, X₂ is threonine, glutamic acid or methionine, X₃ isphenylalanine or tyrosine, X₄ is lysine, threonine, glycine, asparagineor glutamic acid, X₅ is alanine or proline, X₆ is methionine, leucine,valine or isoleucine; (b) the amino acid sequence of HC CDR2 is as setforth in RIRSX₇X₈NX₉YX₁₀TX₁₁YX₁₂DX₁₃VK (SEQ ID NO. 3), X₇ is lysine orglycine, X₈ is tyrosine or serine, X₉ is asparagine or lysine, X₁₀ isalanine or glutamic acid, X₁₁ is tyrosine or glutamic acid, X₁₂ isalanine or lysine, X₁₃ is serine, glutamic acid, aspartic acid, alanine,or glutamine; (c) the amino acid sequence of HC CDR3 is as set forth inHX₁₄NFX₁₅ X₁₆SX₁₇ISYWAX₁₈ (SEQ ID NO. 4), X₁₄ is glycine, alanine, orthreonine, X₁₅ is glycine or asparagine, X₁₆ is asparagine or asparticacid, X₁₇ is tyrosine, histidine, proline, glutamine, leucine orglycine, X₁₈ is tyrosine or threonine; (d) the amino acid sequence of LCCDR1 is as set forth in X₁₉X₂₀X₂₁X₂₂GX₂₃VX₂₄X₂₅GX₂₆YPN (SEQ ID NO. 5),X₁₉ is glycine or alanine, X₂₀ is serine or glutamic acid, X₂₁ is serineor tyrosine, X₂₂ is threonine, phenylalanine, lysine, or serine, X₂₃ isalanine or tyrosine, X₂₄ is threonine or valine, X₂₅ is serine, asparticacid, lysine, histidine or valine,X₂₆ asparagine or tyrosine; (e) theamino acid sequence of LC CDR2 is as set forth in GX₂₇X₂₈X₂₉X₃₀X₃₁P (SEQID NO. 6), X₂₇ is threonine or isoleucine, X₂₈ is lysine, glutamic acid,tyrosine, asparagine or serine, X₂₉ is phenylalanine, leucine, glutamicacid, isoleucine, methionine, or valine, X₃₀ is leucine, asparagine, orglycine, X₃₁ is alanine or valine; and (f) the amino acid sequence of LCCDR3 is as set forth in X₃₂LWYX₃₃NX₃₄WX₃₅ (SEQ ID NO. 7), X₃₂ is valine,threonine or alanine, X₃₃ is serine, aspartic acid or alanine, X₃₄ isarginine or serine, X₃₅ is valine, isoleucine or alanine, wherein X₁,X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇,X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, X₂₆, X₂₇, X₂₈, X₂₉, X₃₀, X₃₁,X₃₂, X₃₃, X₃₄ and X₃₅ are not simultaneously phenylalanine, threonine,phenylalanine, lysine, alanine, methionine, lysine, tyrosine,asparagine, alanine, tyrosine, alanine, serine, glycine, glycine,asparagine, tyrosine, tyrosine, glycine, serine, serine, threonine,alanine, threonine, serine, asparagine, threonine, lysine,phenylalanine, leucine, alanine, valine, serine, arginine, and valinerespectively.

In some embodiments, the single chain variable fragment CD3 bindingprotein comprises the following formula:f1-r1-f2-r2-f3-r3-f4-r4-f5-r5-f6-r6-f7, wherein, r1 is SEQ ID NO: 2; r2is SEQ ID NO: 3; r3 is SEQ ID NO: 4; r4 is SEQ ID NO:5; r5 is SEQ IDNO:6; and r6 is SEQ ID NO:7; and wherein f₁, f₂, f₃, f₄, and f₅ areframework residues selected so that said protein is at least eightypercent identical to the amino acid sequence set forth in SEQ ID NO: 22.

In some embodiments, the single chain variable fragment CD3 bindingprotein comprises an amino acid sequence wherein r1 comprises SEQ ID NO.29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ IDNO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO.38, SEQID NO. 39, or SEQ ID NO. 40. In some embodiments, the single chainvariable fragment CD3 binding protein comprises an amino acid sequencewherein r2 comprises SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ IDNO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQID NO. 49, or SEQ ID NO. 50. In some embodiments, the single chainvariable fragment CD3 binding protein comprises an amino acid sequencewherein r3 comprises SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ IDNO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQID NO. 50, or SEQ ID NO. 60. In some embodiments, the single chainvariable fragment CD3 binding protein comprises an amino acid sequencewherein r4 comprises SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ IDNO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO. 72, or SEQ ID NO. 73In some embodiments, the single chain variable fragment CD3 bindingprotein comprises an amino acid sequence wherein r5 comprises SEQ ID NO.74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ IDNO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQID NO. 84, SEQ ID NO. 85, or SEQ ID NO. 86. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises an aminoacid sequence wherein r6 comprises SEQ ID NO. 87, SEQ ID NO. 88, SEQ IDNO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, or SEQ ID NO. 93.In some embodiments, the single chain variable fragment CD3 bindingprotein comprises an amino acid sequence wherein r1 is SEQ ID NO. 39, r2is SEQ ID NO. 49, r3 is SEQ ID NO. 51, r4 is SEQ ID NO. 61, r5 is SEQ IDNO. 86, and r6 is SEQ ID NO. 87. In some embodiments, the single chainvariable fragment CD3 binding protein comprises an amino acid sequencewherein r1 is SEQ ID NO. 30, r2 is SEQ ID NO. 43, r4 is SEQ ID NO. 64,and r6 is SEQ ID NO. 89. In some embodiments, the single chain variablefragment CD3 binding protein comprises an amino acid sequence wherein r3is SEQ ID NO. 55, r4 is SEQ ID NO. 67, r5 is SEQ ID NO. 77, and r6 isSEQ ID NO. 92. In some embodiments, the single chain variable fragmentCD3 binding protein comprises an amino acid sequence wherein r1 is SEQID NO. 31, r2 is SEQ ID NO. 42, r3 is SEQ ID NO. 60, r4 is SEQ ID NO.64, r5 is SEQ ID NO. 79, and r6 is SEQ ID NO. 91. In some embodiments,the single chain variable fragment CD3 binding protein comprises anamino acid sequence wherein r1 is SEQ ID NO. 35, r2 is SEQ ID NO. 46, r3is SEQ ID NO. 56, r4 is SEQ ID NO. 68, and r5 is SEQ ID NO. 75. In someembodiments, the single chain variable fragment CD3 binding proteincomprises an amino acid sequence wherein r1 is SEQ ID NO. 32, r2 is SEQID NO. 47, r3 is SEQ ID NO. 56, r4 is SEQ ID NO. 65, r5 is SEQ ID NO.80, and r6 is SEQ ID NO. 87. In some embodiments, the single chainvariable fragment CD3 binding protein comprises an amino acid sequencewherein r1 is SEQ ID NO. 29, r2 is SEQ ID NO. 44, r3 is SEQ ID NO. 52,r4 is SEQ ID NO. 73, and r5 is SEQ ID NO. 76. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises an aminoacid sequence wherein r1 is SEQ ID NO. 33, r2 is SEQ ID NO. 48, r3 isSEQ ID NO. 57, r4 is SEQ ID NO. 69, and r5 is SEQ ID NO. 74. In someembodiments, the single chain variable fragment CD3 binding proteincomprises an amino acid sequence wherein r1 is SEQ ID NO. 38, r4 is SEQID NO. 62, and r5 is SEQ ID NO. 81. In some embodiments, the singlechain variable fragment CD3 binding protein comprises an amino acidsequence wherein r1 is SEQ ID NO. 37, r3 is SEQ ID NO. 53, r4 is SEQ IDNO. 70, r5 is SEQ ID NO. 82, and r6 is SEQ ID NO. 88. In someembodiments, the single chain variable fragment CD3 binding proteincomprises an amino acid sequence wherein r1 is SEQ ID NO. 34, r2 is SEQID NO. 47, r3 is SEQ ID NO. 56, r4 is SEQ ID NO. 68 and r5 is SEQ ID NO.75. In some embodiments, the single chain variable fragment CD3 bindingprotein comprises an amino acid sequence wherein r1 is SEQ ID NO. 29, r3is SEQ ID NO. 54, r4 is SEQ ID NO. 71 and r5 is SEQ ID NO. 83. In someembodiments, the single chain variable fragment CD3 binding proteincomprises an amino acid sequence wherein r1 is SEQ ID NO. 33, r2 is SEQID NO. 41, r4 is SEQ ID NO. 63, r5 is SEQ ID NO. 84 and r6 is SEQ ID NO.90. In some embodiments, the single chain variable fragment CD3 bindingprotein comprises an amino acid sequence wherein r1 is SEQ ID NO. 30, r2is SEQ ID NO. 44, r3 is SEQ ID NO. 58, r4 is SEQ ID NO. 66 and r5 is SEQID NO. 85. In some embodiments, the single chain variable fragment CD3binding protein comprises an amino acid sequence wherein r1 is SEQ IDNO. 40, r2 is SEQ ID NO. 45, r3 is SEQ ID NO. 56, r5 is SEQ ID NO. 78and r6 is SEQ ID NO. 93. In some embodiments, the single chain variablefragment CD3 binding protein comprises an amino acid sequence wherein r1is SEQ ID NO. 36, r2 is SEQ ID NO. 50, r3 is SEQ ID NO. 59, r4 is SEQ IDNO. 72 and r5 is SEQ ID NO. 75.

In some embodiments, the single chain variable fragment CD3 bindingprotein has an amino acid sequence selected from SEQ ID NO. 8, SEQ IDNO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18,SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 94, and SEQ IDNO. 95. In some embodiments, the single chain variable fragment CD3binding protein has the amino acid sequence set forth as SEQ ID NO. 8.In some embodiments, the single chain variable fragment CD3 bindingprotein has the amino acid sequence set forth as SEQ ID NO. 9. In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 14. In some embodiments,the single chain variable fragment CD3 binding protein has the aminoacid sequence set forth as SEQ ID NO. 19. In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 94. In some embodiments, the singlechain variable fragment CD3 binding protein comprises an amino acidsequence comprising a linker, wherein said linker comprises the aminoacid sequence as set forth in GGGGSGGGGSGGGGS (SEQ ID NO: 1). In someembodiments, the single chain variable fragment CD3 binding proteinbinds to CD3 selected from human CD3 and cynomolgus CD3. In someembodiments, the single chain variable fragment CD3 binding proteinbinds to human CD3 and cynomolgus CD3 with comparable binding affinity(Kd). In some embodiments, the single chain variable fragment CD3binding protein binds to human CD3 with a human Kd (hKd) between about 1nM and about 200 nM and to cynomolgus CD3 with a cynomolgus Kd (cKd)between about 1 nM and about 300 nM. In some embodiments, the hKd andthe cKd are between about 3 nM to about 5 nM, about 6 nM to about 10 nM,about 11 nM to about 20 nM, about 25 nM to about 40 nM,about 40 nM toabout 60 nM, about 70 nM to about 90 nM, about 100 nM to about 120 nM,about 125 nM to about 140 nM, about 145 nM to about 160 nM, about 170 nMand to about 200 nM, about 210 nM to about 250 nM, about 260 nM to about300 nM.

In some embodiments, the single chain variable fragment CD3 bindingprotein binds to human CD3 with a human Kd (hKd), binds to cynomolgusCD3 with a cynomolgus Kd (cKd), and the hKd and the cKd are about thesame as the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteinbinds to human CD3 with a human Kd (hKd), binds to cynomolgus CD3 with acynomolgus Kd (cKd), and the hKd and the cKd are between about 1.5-foldto about 2-fold higher than the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein binds to human CD3 with a human Kd (hKd), binds to cynomolgusCD3 with a cynomolgus Kd (cKd), and the hKd and the cKd are betweenabout 3-fold to about 5-fold higher than the binding affinity towardsCD3 of a protein which has the sequence as set forth in wt anti-CD3 (SEQID NO.22). In some embodiments, the single chain variable fragment CD3binding protein binds to human CD3 with a human Kd (hKd), binds tocynomolgus CD3 with a cynomolgus Kd (cKd), and the hKd and the cKd arebetween about 6-fold to about 15-fold higher than the binding affinitytowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variablefragment CD3 binding protein binds to human CD3 with a human Kd (hKd),binds to cynomolgus CD3 with a cynomolgus Kd (cKd), and the hKd and thecKd are between about 20-fold to about 50-fold higher than the bindingaffinity towards CD3 of a protein which has the sequence as set forth inwt anti-CD3 (SEQ ID NO.22).

In some embodiments, the single chain variable fragment CD3 bindingprotein has the amino acid sequence set forth as SEQ ID NO. 8, and thehKd and cKd are about the same as the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein has the amino acid sequence set forth as SEQ ID NO. 8, and thehKd and the cKd are between about 3 nM and about 5 nM. In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 9, and the hKd and thecKd are about the same as the binding affinity towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteinhas the amino acid sequence set forth as SEQ ID NO. 9, and the hKd andthe cKd are between about 3 nM and about 5 nM. In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 10, and the hKd and cKd are about thesame as the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 10, and the hKd and thecKd are between about 3 nM and about 5 nM. In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 11, and wherein the hKd and cKd areabout about the same as the binding affinity towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteinhas the amino acid sequence set forth as SEQ ID NO. 11, and the hKd andthe cKd are between about 3 nM and about 5 nM. In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 12, and the hKd and cKd are betweenabout 1.5-fold to about 2-fold higher than the binding affinity towardsCD3 of a protein which has the sequence as set forth in wt anti-CD3 (SEQID NO. 22). In some embodiments, the single chain variable fragment CD3binding protein has the amino acid sequence set forth as SEQ ID NO. 12,and the hKd and the cKd are between about 6 nM and about 10 nM. In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 13, and the hKd and thecKd are between about 1.5-fold to about 2-fold higher than the bindingaffinity towards CD3 of a protein which has the sequence as set forth inwt anti-CD3 (SEQ ID NO. 22). In some embodiments, the single chainvariable fragment CD3 binding protein has the amino acid sequence setforth as SEQ ID NO. 13, and the hKd and the cKd are between about 6 nMand about 10 nM. In some embodiments, the single chain variable fragmentCD3 binding protein has the amino acid sequence set forth as SEQ ID NO.14, and the hKd and the cKd are about 3-fold to about 5-fold higher thanthe binding affinity towards CD3 of a protein which has the sequence asset forth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 14, and the hKd and the cKd are betweenabout 11 nM and 20 nM. In some embodiments, the single chain variablefragment CD3 binding protein has the amino acid sequence set forth asSEQ ID NO. 15, and the hKd and cKd are about 6-fold to about 15-foldhigher than the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 15, and the hKd and thecKd are between about 25 nM and about 40 nM. In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 16, and the hKd and the cKd are about3-fold to about 5-fold higher than the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein has the amino acid sequence set forth as SEQ ID NO. 16, and thehKd iand the cKd are between about 11 nM and 20 nM. In some embodiments,the single chain variable fragment CD3 binding protein has the aminoacid sequence set forth as SEQ ID NO. 17, and the hKd and cKd are about6-fold to about 15-fold higher than the binding affinity towards CD3 ofa protein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein has the amino acid sequence set forth as SEQ ID NO. 17, and thehKd and the cKd are between about 40 nM and about 60 nM. In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 18, and the hKd and cKdare about 3-fold to about 5-fold higher than the binding affinitytowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein has the amino acid sequence set forth asSEQ ID NO. 18, and the hKd and the cKd are between about 11 nM and 20nM. In some embodiments, the single chain variable fragment CD3 bindingprotein has the amino acid sequence set forth as SEQ ID NO. 19, and thehKd and cKd are about 6-fold to about 15-fold higher than the bindingaffinity towards CD3 of a protein which has the sequence as set forth inwt anti-CD3 (SEQ ID NO. 22). In some embodiments, the single chainvariable fragment CD3 binding protein has the amino acid sequence setforth as SEQ ID NO. 19, and the hKd and the cKd are between about 40 nMand 60 nM. In some embodiments, the single chain variable fragment CD3binding protein has the amino acid sequence set forth as SEQ ID NO. 20,and the hKd and the cKd are about 3-fold to about 5-fold higher than thebinding affinity towards CD3 of a protein which has the sequence as setforth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, the singlechain variable fragment CD3 binding protein has the amino acid sequenceset forth as SEQ ID NO. 20, and the hKd and the cKd are between about 11nM and about 20 nM. In some embodiments, the single chain variablefragment CD3 binding protein has the amino acid sequence set forth asSEQ ID NO. 21, and the hKd and the cKd are about 20-fold to about50-fold higher than the binding affinity towards CD3 of a protein whichhas the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasthe amino acid sequence set forth as SEQ ID NO. 21, and the hKd and thecKd are between about 125 nM and about 140 nM. In some embodiments, thesingle chain variable fragment CD3 binding protein has the amino acidsequence set forth as SEQ ID NO. 94, and wherein the hKd and the cKd areabout 20-fold to about 50-fold higher than the binding affinity towardsCD3 of a protein which has the sequence as set forth in wt anti-CD3 (SEQID NO. 22). In some embodiments, the single chain variable fragment CD3binding protein has the amino acid sequence set forth as SEQ ID NO. 94,and the hKd and the cKd are between about 100 nM and about 120 nM. Insome embodiments, the single chain variable fragment CD3 binding proteinhas the amino acid sequence set forth as SEQ ID NO. 95, and the hKd andthe cKd are about 20-fold to about 50-fold higher than the bindingaffinity towards CD3 of a protein which has the sequence as set forth inwt anti-CD3 (SEQ ID NO. 22). In some embodiments, the single chainvariable fragment CD3 binding protein has the amino acid sequence setforth as SEQ ID NO. 95, and the hKd and the cKd are between about 100 nMand about 120 nM.

Provided herein in another embodiment is a single chain variablefragment CD3 binding protein comprising the sequence set forth as SEQ IDNO. 22 (wt anti-CD3) wherein one or more amino acid residues selectedfrom amino acid positions 27, 28, 29, 31, 33, 34, 54, 55, 57, 59, 61,63, 65, 102, 105, 106, 108, 114, 163, 164, 165, 166, 168, 170, 171, 173,193, 194, 195, 196, 197, 231, 235, 237, and 239 are substituted, whereinamino acid position 27 is substituted with asparagine, amino acidposition 28 is substituted with glutamic acid, or methionine,amino acidposition 29 is substituted with tyrosine, amino acid position 31 issubstituted with asparagine, glycine, glutamic acid or threonine, aminoacid position 33 is substituted with proline, amino acid position 34 issubstituted with valine, leucine or isoleucine, amino acid position 54is substituted with glycine, amino acid position 55 is substituted withserine, amino acid position 57 is substituted with lysine, amino acidposition 59 is substituted with glutamic acid, amino acid position 61 issubstituted with glutamic acid, amino acid position 63 is substitutedwith lysine, amino acid position 65 is substituted with aspartic acid,glutamic acid, alanine, or glutamine, amino acid position 102 issubstituted with alanine or threonine, amino acid position 105 issubstituted with asparagine, amino acid position 106 is substituted withaspartic acid, amino acid position 108 is substituted with histidine,proline, glutamine, glycine, or leucine, amino acid position 114 issubstituted with threonine, amino acid position 163 is substituted withalanine,amino acid position 164 is substituted with glutamic acid, aminoacid position 165 is substituted with tyrosine, amino acid position 166is substituted with phenylalanine, lysine, or serine, amino acidposition 168 is substituted with tyrosine, amino acid position 170 issubstituted with valine, amino acid position 171 is substituted withaspartic acid, lysine, valine, or histidine, amino acid position 173 issubstituted with tyrosine, amino acid position 193 is substituted withisoleucine, amino acid position 194 is substituted with glutamic acid,tyrosine, asparagine, or serine, amino acid position 195 is substitutedwith leucine, glutamic acid, isoleucine, methionine, or valine, aminoacid position 196 is substituted with asparagine, or glycine, amino acidposition 197 is substituted with valine, amino acid position 231 issubstituted with threonine, or alanine, amino acid position 235 issubstituted with aspartic acid, or alanine, amino acid position 237 issubstituted with serine, and amino acid position 239 is substituted withalanine, or isoleucine. In some embodiments, the single chain variablefragment CD3 binding protein comprises one or more additionalsubstitutions in amino acid positions other than positions 27, 28, 29,31, 33, 34, 54, 55, 57, 59, 61, 63, 65, 102, 105, 106, 108, 114, 163,164, 165, 166, 168, 170, 171, 173, 193, 194, 195, 196, 197, 231, 235,237, and 239. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a substitution in position 27. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 28. In some embodiments, the singlechain variable fragment CD3 binding protein comprises a substitution inposition 29. In some embodiments, the single chain variable fragment CD3binding protein comprises a substitution in position 31. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 33. In some embodiments, the singlechain variable fragment CD3 binding protein comprises a substitution inposition 34. In some embodiments, the single chain variable fragment CD3binding protein comprises a substitution in position 54. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 55. In some embodiments, the singlechain variable fragment CD3 binding protein comprises a substitution inposition 57. In some embodiments, the single chain variable fragment CD3binding protein comprises a substitution in position 59. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 61. In some embodiments, the singlechain variable fragment CD3 binding protein comprises a substitution inposition 63. In some embodiments, the single chain variable fragment CD3binding protein comprises a substitution in position 65. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 102. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises asubstitution in position 105. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a substitution inposition 106. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a substitution in position 108. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 114. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises asubstitution in position 163. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a substitution inposition 164. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a substitution in position 165. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 166. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises asubstitution in position 168. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a substitution inposition 170. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a substitution in position 171. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 173. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises asubstitution in position 193. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a substitution inposition 194. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a substitution in position 195. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 196. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises asubstitution in position 197. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a substitution inposition 231. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a substitution in position 235. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a substitution in position 237. In some embodiments, thesingle chain variable fragment CD3 binding protein comprises asubstitution in position 239. In some embodiments, the single chainvariable fragment CD3 binding protein comprises substitutions inpositions 34, 65, 102, 163, 197, and 231. In some embodiments, thesingle chain variable fragment CD3 binding protein comprisessubstitutions in positions 28, 57, 166, and 235. In some embodiments,the single chain variable fragment CD3 binding protein comprisessubstitutions in positions 108, 168, 194, and 239. In some embodiments,the single chain variable fragment CD3 binding protein comprisessubstitutions in positions 28, 55, 114, 166, 195, and 237. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 31, 63, 108, 170, and 194. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 29, 65, 108, 166, 195, and 231. Insome embodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 27, 59, 102, 173, and 194. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 31, 65, 108, 171, and 193. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 34, 164, and 195. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 33, 105, 171, 195, and 231. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 31, 65, 108, 170, and 194. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 27, 106, 171, and 195. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 31, 54, 165, 196, and 235. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 28, 59, 108, 166, and 196. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 34, 61, 108, 194, and 239. In someembodiments, the single chain variable fragment CD3 binding proteincomprises substitutions in positions 31, 65, 108, 171, and 194.

In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid positions 34, 65, 102,163, 197, and 231 are substituted, and wherein the hKd and the cKd areabout the same as the binding affinity towards CD3 of a protein whichhas the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 34, 65, 102, 163, 197,and 231 are substituted, and wherein the hKd and the cKd are betweenabout 3 nM and about 5 nM. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a sequence wherein aminoacid positions 28, 57, 166, and 235 are substituted, and wherein the hKdand the cKd are about the same as the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid positions 28, 57, 166,and 235 are substituted, and wherein the hKd and the cKd are betweenabout 3 nM and about 5 nM.

In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid positions 108, 168, 194,and 239 are substituted, and wherein the hKd and the cKd are about thesame as the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 108, 168, 194, and 239are substituted, and wherein the hKd and the cKd are between about 3 nMand about 5 nM. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a sequence wherein amino acid positions28, 55, 114, 166, 195, and 237 are substituted, and wherein the hKd andthe cKd are about the same as the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid positions 28, 55, 114,166, 195, and 237 are substituted, and wherein the hKd and the cKd arebetween about 3 nM and about 5 nM. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a sequence wherein aminoacid positions 31, 63, 108, 170, and 194 are substituted, and whereinthe hKd and the cKd are between about 1.5-fold to about 2-fold higherthan the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 31, 63, 108, 170, and194 are substituted, and wherein the hKd and the cKd are between about 6nM and about 10 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidpositions 29, 65, 108, 166, 195, and 231 are substituted, and whereinthe hKd and the cKd are between about 1.5-fold to about 2-fold higherthan the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 29, 65, 108, 166, 195,and 231 are substituted, and wherein the hKd and the cKd are betweenabout 6 nM and about 10 nM. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a sequence wherein aminoacid positions 27, 59, 102, 173, and 194 are substituted, and whereinthe hKd and the cKd are about 3-fold to about 5-fold higher than thebinding affinity towards CD3 of a protein which has the sequence as setforth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, the singlechain variable fragment CD3 binding protein comprises a sequence whereinamino acid positions 27, 59, 102, 173, and 194 are substituted, andwherein the hKd and the cKd are between about 11 nM and 20 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 31, 65, 108, 171, and193 are substituted, and wherein the hKd and the cKd are about 6-fold toabout 15-fold higher than the binding affinity towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 31, 65, 108, 171, and193 are substituted, and wherein the hKd and the cKd are between about25 nM and about 40 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidpositions 34, 164, and 195 are substituted, and wherein the hKd and thecKd are about 3-fold to about 5-fold higher than the binding affinitytowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidpositions 34, 164, and 195 are substituted, and wherein the hKd and thecKd are between about 11 nM and 20 nM. In some embodiments, the singlechain variable fragment CD3 binding protein comprises a sequence whereinamino acid positions 33, 105, 171, 195, and 231 are substituted, andwherein the hKd and the cKd are about 6-fold to about 15-fold higherthan the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 33, 105, 171, 195, and231 are substituted, and wherein the hKd and the cKd are between about40 nM and about 60 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidpositions 31, 65, 108, 170, and 194 are substituted, and wherein the hKdand the cKd are about 3-fold to about 5-fold higher than the bindingaffinity towards CD3 of a protein which has the sequence as set forth inwt anti-CD3 (SEQ ID NO. 22). In some embodiments, the single chainvariable fragment CD3 binding protein comprises a sequence wherein aminoacid positions 31, 65, 108, 170, and 194 are substituted, and whereinthe hKd and the cKd are between about 11 nM and 20 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 27, 106, 171, and 195are substituted, and wherein the hKd and the cKd are about 6-fold toabout 15-fold higher than the binding affinity towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 27, 106, 171, and 195are substituted, and wherein the hKd and the cKd are between about 40 nMand 60 nM. In some embodiments, the single chain variable fragment CD3binding protein comprises a sequence wherein amino acid positions 31,54, 165, 196, and 235 are substituted, and wherein the hKd and the cKdare about 3-fold to about 5-fold higher than the binding affinitytowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidpositions 31, 54, 165, 196, and 235 are substituted (10B2), and whereinthe hKd and the cKd are between about 11 nM and about 20 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 28, 59, 108, 166, and196 are substituted, and wherein the hKd and the cKd are about 25-foldto about 50-fold higher than the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid positions 28, 59, 108,166, and 196 are substituted, and wherein the hKd and the cKd arebetween about 125 nM and about 140 nM. In some embodiments, the singlechain variable fragment CD3 binding protein comprises a sequence whereinamino acid positions 34, 61, 108, 194, and 239 are substituted, andwherein the hKd and the cKd are about 20-fold to about 50-fold higherthan the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid positions 34, 61, 108, 194, and239 are substituted, and wherein the hKd and the cKd are between about100 nM and about 120 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidpositions 31, 65, 108, 171, and 194 are substituted, and wherein the hKdand the cKd are about 20-fold to about 50-fold higher than the bindingaffinity towards CD3 of a protein which has the sequence as set forth inwt anti-CD3 (SEQ ID NO. 22). In some embodiments, the single chainvariable fragment CD3 binding protein comprises a sequence wherein aminoacid positions 31, 65, 108, 171, and 194 are substituted, and whereinthe hKd and the cKd are between about 100 nM and about 120 nM.

Provided herein in a further embodiment is a single chain variablefragment CD3 binding protein comprising an amino acid sequence as setforth in wt anti-CD3 (SEQ ID NO: 22), comprising a variable heavy chainregion (VH), a variable light chain region (VL), a linker comprising theamino acid sequence as set forth in GGGGSGGGGSGGGGS (SEQ ID NO: 1),wherein VH comprises complementarity determining regions CDR1, CDR2, andCDR3, wherein VL comprises complementarity determining regions LC CDR1,LC CDR2, and LC CDR3, comprising at least one mutation in CDR1, CDR2 orCDR3 of VH, and LC CDR1, LC CDR2 or LC CDR3 of VL, wherein the at leastone mutation is not in amino acid positions 26, 30, 32, 35, 50, 51, 52,53, 56, 58, 60, 62, 64, 66, 67, 101, 103, 104, 107, 109, 110, 111, 112,113, 167, 169, 172, 174, 175, 176, 192, 198, 232, 233, 234, 236, or 238.In some embodiments, the single chain variable fragment CD3 bindingprotein comprises at least one mutation in amino acid position selectedfrom 27, 28, 29, 31, 33, 34, 54, 55, 57, 59, 61, 63, 65, 102, 105, 106,108, 114, 163, 164, 165, 166, 168, 170, 171, 173, 193, 194, 195, 196,197, 231, 235, 237, and 239. In some embodiments, amino acid position 34is mutated to isoleucine, position 65 is mutated to glutamine, position102 is mutated to alanine, position 163 is mutated to alanine, position197 is mutated to valine, and position 231 is mutated to threonine. Insome embodiments, amino acid position 28 is mutated to glutamic acid,position 57 is mutated to lysine, position 166 is mutated tophenylalanine, and position 235 is mutated to aspartic acid. In someembodiments, amino acid position 108 is mutated to histidine, position168 is mutated to tyrosine, position 194 is mutated to serine, andposition 239 is mutated to isoleucine. In some embodiments, amino acidposition 28 is mutated to methionine, position 55 is mutated to serine,position 114 is mutated to threonine, position 166 is mutated tophenylalanine, position 195 is mutated to leucine, and position 237 ismutated to serine. In some embodiments, amino acid position 31 ismutated to threonine, position 63 is mutated to lysine, position 108 ismutated to proline, position 170 is mutated to valine, and position 194is mutated to glutamic acid. In some embodiments, amino acid position 29is mutated to tyrosine, position 65 is mutated to glutamic acid,position 108 is mutated to proline, position 166 is mutated to lysine,position 195 is mutated to glutamic acid, and position 231 is mutated tothreonine. In some embodiments, amino acid position 27 is mutated toasparagine, position 59 is mutated to glutamic acid, position 102 ismutated to threonine, position 173 is mutated to tyrosine, and position194 is mutated to tyrosine. In some embodiments, amino acid positionamino acid position 31 is mutated to asparagine, position 65 is mutatedto alanine, position 108 is mutated to glutamine, position 171 ismutated to aspartic acid, and position 193 is mutated to isoleucine. Insome embodiments, amino acid position 34 is mutated to valine, position164 is mutated to glutamic acid, and position 195 is mutated toisoleucine. In some embodiments, amino acid position 33 is mutated toproline, position 105 is mutated to asparagine, position 171 is mutatedto lysine, position 195 is mutated to methionine, and position 231 ismutated to alanine. In some embodiments, amino acid position 31 ismutated to glycine, position 65 is mutated to glutamic acid, position108 is mutated to proline, position 170 is mutated to valine, andposition 194 is mutated to glutamic acid. In some embodiments, aminoacid position 27 is mutated to asparagine, position 106 is mutated toaspartic acid, position 171 is mutated to histidine, and position 195 ismutated to valine. In some embodiments, amino acid position 31 ismutated to asparagine, position 54 is mutated to glycine, position 165is mutated to tyrosine, position 196 is mutated to asparagine, andposition 235 is mutated to alanine. In some embodiments, amino acidposition 28 is mutated to glutamic acid, position 59 is mutated toglutamic acid, position 108 is mutated to leucine, position 166 ismutated to serine, and position 196 is mutated to glycine. In someembodiments, amino acid position 34 is substituted with leucine, aminoacid position 61 is substituted with glutamic acid, amino acid position108 is substituted with proline, amino acid position 194 is substitutedwith asparagine, and amino acid position 239 is substituted withalanine. In some embodiments, amino acid position 31 is substituted withglutamic acid, amino acid position 65 is substituted with aspartic acid,amino acid position 108 is substituted with glycine, amino acid position171 is substituted with valine, and amino acid position 194 issubstituted with glutamic acid.

In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid position 34 is mutatedto isoleucine, position 65 is mutated to glutamine, position 102 ismutated to alanine, position 163 is mutated to alanine, position 197 ismutated to valine, and position 231 is mutated to threonine, and whereinthe hKd and the cKd are about the same as the binding affinity towardsCD3 of a protein which has the sequence as set forth in wt anti-CD3 (SEQID NO. 22). In some embodiments, the single chain variable fragment CD3binding protein comprises a sequence wherein amino acid position 34 ismutated to isoleucine, position 65 is mutated to glutamine, position 102is mutated to alanine, position 163 is mutated to alanine, position 197is mutated to valine, and position 231 is mutated to threonine, whereinthe hKd and the cKd are between about 3 nM and 5 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 28 is mutated toglutamic acid, position 57 is mutated to lysine, position 166 is mutatedto phenylalanine, and position 235 is mutated to aspartic acid, andwherein the hKd and the cKd are about the same as the binding affinitytowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidposition 28 is mutated to glutamic acid, position 57 is mutated tolysine, position 166 is mutated to phenylalanine, and position 235 ismutated to aspartic acid, wherein the hKd and the cKd are between about3 nM and about 5 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidposition 108 is mutated to histidine, position 168 is mutated totyrosine, position 194 is mutated to serine, and position 239 is mutatedto isoleucine, and wherein the hKd and the cKd are about the same as thebinding affinity towards CD3 of a protein which has the sequence as setforth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, the singlechain variable fragment CD3 binding protein comprises a sequence whereinamino acid position 108 is mutated to histidine, position 168 is mutatedto tyrosine, position 194 is mutated to serine, and position 239 ismutated to isoleucine, wherein the hKd and the cKd are between about 3nM and about 5 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidposition 28 is mutated to methionine, position 55 is mutated to serine,position 114 is mutated to threonine, position 166 is mutated tophenylalanine, position 195 is mutated to leucine, and position 237 ismutated to serine, and wherein the hKd and the cKd are about the same asthe binding affinity towards CD3 of a protein which has the sequence asset forth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, thesingle chain variable fragment CD3 binding protein comprises a sequencewherein amino acid position 28 is mutated to methionine, position 55 ismutated to serine, position 114 is mutated to threonine, position 166 ismutated to phenylalanine, position 195 is mutated to leucine, andposition 237 is mutated to serine, wherein the hKd and the cKd arebetween about 3 nM and about 5 nM. In some embodiments, the single chainvariable fragment CD3 binding protein comprises a sequence wherein aminoacid position 31 is mutated to threonine, position 63 is mutated tolysine, position 108 is mutated to proline, position 170 is mutated tovaline, and position 194 is mutated to glutamic acid, and wherein thehKd and the cKd are between about 1.5-fold to about 2-fold higher thanthe binding affinity towards CD3 of a protein which has the sequence asset forth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, thesingle chain variable fragment CD3 binding protein comprises a sequencewherein amino acid position 31 is mutated to threonine, position 63 ismutated to lysine, position 108 is mutated to proline, position 170 ismutated to valine, and position 194 is mutated to glutamic acid, whereinthe hKd and the cKd are between about 6 nM and about 10 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 29 is mutated totyrosine, position 65 is mutated to glutamic acid, position 108 ismutated to proline, position 166 is mutated to lysine, position 195 ismutated to glutamic acid, and position 231 is mutated to threonine, andwherein the hKd and the cKd are between about 1.5-fold to about 2-foldhigher than the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 29 is mutated totyrosine, position 65 is mutated to glutamic acid, position 108 ismutated to proline, position 166 is mutated to lysine, position 195 ismutated to glutamic acid, and position 231 is mutated to threonine,wherein the hKd and the cKd are between about 6 nM and about 10 nM. Insome embodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 27 is mutated toasparagine, position 59 is mutated to glutamic acid, position 102 ismutated to threonine, position 173 is mutated to tyrosine, and position194 is mutated to tyrosine, and wherein the hKd and the cKd are about3-fold to about 5-fold higher than the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid position 27 is mutatedto asparagine, position 59 is mutated to glutamic acid, position 102 ismutated to threonine, position 173 is mutated to tyrosine, and position194 is mutated to tyrosine, wherein the hKd and the cKd are betweenabout 11 nM and 20 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidposition 31 is mutated to asparagine, position 65 is mutated to alanine,position 108 is mutated to glutamine, position 171 is mutated toaspartic acid, and position 193 is mutated to isoleucine, and whereinthe hKd and the cKd are about 6-fold to about 15-fold higher than thebinding affinity towards CD3 of a protein which has the sequence as setforth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, the singlechain variable fragment CD3 binding protein comprises a sequence whereinamino acid position 31 is mutated to asparagine, position 65 is mutatedto alanine, position 108 is mutated to glutamine, position 171 ismutated to aspartic acid, and position 193 is mutated to isoleucine,wherein the hKd and the cKd are between about 25 nM and about 40 nM. Insome embodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 34 is mutated tovaline, position 164 is mutated to glutamic acid, and position 195 ismutated to isoleucine, and wherein the hKd and the cKd are about 3-foldto about 5-fold higher than the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid position 34 is mutatedto valine, position 164 is mutated to glutamic acid, and position 195 ismutated to isoleucine, wherein the hKd and the cKd are between about 11nM and 20 nM. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a sequence wherein amino acid position 33is mutated to proline, position 90 is mutated to asparagine, position105 is mutated to asparagine, position 171 is mutated to lysine,position 195 is mutated to methionine, and position 231 is mutated toalanine, and wherein the hKd and the cKd are about 6-fold to about15-fold higher than the binding affinity towards CD3 of a protein whichhas the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 33 is mutated toproline, position 90 is mutated to asparagine, position 105 is mutatedto asparagine, position 171 is mutated to lysine, position 195 ismutated to methionine, and position 231 is mutated to alanine, whereinthe hKd and the cKd are between about 40 nM and 60 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 31 is mutated toglycine, position 65 is mutated to glutamic acid, position 108 ismutated to proline, position 170 is mutated to valine, and position 194is mutated to glutamic acid, and wherein the hKd and the cKd are about3-fold to about 5-fold higher than the binding affinity towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein comprises a sequence wherein amino acid position 31 is mutatedto glycine, position 65 is mutated to glutamic acid, position 108 ismutated to proline, position 170 is mutated to valine, and position 194is mutated to glutamic acid, wherein the hKd and the cKd are betweenabout 11 nM and 20 nM. In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidposition 27 is mutated to asparagine, position 106 is mutated toaspartic acid, position 171 is mutated to histidine, and position 195 ismutated to valine, and wherein the hKd and the cKd are about 6-fold toabout 15-fold higher than the binding affinity towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 27 is mutated toasparagine, position 106 is mutated to aspartic acid, position 171 ismutated to histidine, and position 195 is mutated to valine, wherein thehKd and the cKd are between about 40 nM and 60 nM. In some embodiments,the single chain variable fragment CD3 binding protein comprises asequence wherein amino acid position 31 is mutated to asparagine,position 54 is mutated to glycine, position 165 is mutated to tyrosine,position 196 is mutated to asparagine, and position 235 is mutated toalanine, and wherein the hKd and the cKd are about 3-fold to about5-fold higher than the binding affinity towards CD3 of a protein whichhas the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 31 is mutated toasparagine, position 54 is mutated to glycine, position 165 is mutatedto tyrosine, position 196 is mutated to asparagine, and position 235 ismutated to alanine, wherein the hKd and the cKd are between about 11 nMand about 20 nM. In some embodiments, the single chain variable fragmentCD3 binding protein comprises a sequence wherein amino acid position 28is mutated to glutamic acid, position 59 is mutated to glutamic acid,position 108 is mutated to leucine, position 166 is mutated to serine,and position 196 is mutated to glycine, and wherein the hKd and the cKdare about 20-fold to about 50-fold higher than the binding affinitytowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein comprises a sequence wherein amino acidposition 28 is mutated to glutamic acid, position 59 is mutated toglutamic acid, position 108 is mutated to leucine, position 166 ismutated to serine, and position 196 is mutated to glycine, wherein thehKd and the cKd are between about 125 nM and about 140 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 34 is mutated toleucine, amino acid position 61 is mutated to glutamic acid, amino acidposition 108 is mutated to proline, amino acid position 194 is mutatedto asparagine, amino acid position 239 is mutated to alanine, andwherein the hKd and the cKd are about 20-fold to about 50-fold higherthan the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 34 is mutated toleucine, amino acid position 61 is mutated to glutamic acid, amino acidposition 108 is mutated to proline, amino acid position 194 is mutatedto asparagine, amino acid position 239 is mutated to alanine, whereinthe hKd and the cKd are between about 100 nM and about 120 nM. In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 31 is mutated toglutamic acid, amino acid position 65 is mutated to aspartic acid, aminoacid position 108 is mutated to glycine, amino acid position 171 ismutated to valine, and amino acid position 194 is mutated to glutamicacid, and wherein the hKd and the cKd are about 20-fold to about 50-foldhigher than the binding affinity towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding proteincomprises a sequence wherein amino acid position 31 is mutated toglutamic acid, amino acid position 65 is mutated to aspartic acid, aminoacid position 108 is mutated to glycine, amino acid position 171 ismutated to valine, and amino acid position 194 is mutated to glutamicacid, and wherein the hKd and the cKd are between about 100 nM and about120 nM.

In some embodiments, the single chain variable fragment CD3 bindingprotein does not bind to mouse CD3.

In one embodiment is provided a polynucleotide encoding a single chainvariable fragment CD3 binding protein according to the presentdisclosure. In one embodiment is provided a vector comprising thepolynucleotide described herein. In one embodiment is provided a hostcell transformed with the vector described herein. In a furtherembodiment is provided a pharmaceutical composition comprising (i) asingle chain variable fragment CD3 binding protein according to thepresent disclosure, the polynucleotide according to present disclosure,the vector according to present disclosure or the host cell according topresent disclosure, and (ii) a pharmaceutically acceptable carrier.

In another embodiment is provided a process for the production of asingle chain variable fragment CD3 binding protein according to thepresent disclosure, said process comprising culturing a host transformedor transfected with a vector comprising a nucleic acid sequence encodingsingle chain variable fragment CD3 binding protein according to thepresent disclosure under conditions allowing the expression of thesingle chain variable fragment CD3 binding protein and recovering andpurifying the produced protein from the culture.

In another embodiment is provided a method for the treatment oramelioration of a proliferative disease, a tumorous disease, aninflammatory disease, an immunological disorder, an autoimmune disease,an infectious disease, a viral disease, an allergic reaction, aparasitic reaction, a graft-versus-host disease or a host-versus-graftdisease comprising the administration of the single chain variablefragment CD3 binding protein according to the present disclosure, to asubject in need thereof. In some embodiments, the subject is human. Insome embodiments, the method further comprises administration of anagent in combination with the single chain variable fragment CD3 bindingprotein according to the present disclosure.

Provided herein in one embodiment is a multispecific binding proteincomprising the single chain variable fragment CD3 binding proteinaccording the present disclosure. One embodiment describes an antibodycomprising the single chain variable fragment CD3 binding proteinaccording to the present disclosure. A further embodiment provides amultispecific antibody, a bispecific antibody, a single domain antibody,a variable heavy domain, a peptide, or a ligand, comprising the singlechain variable fragment CD3 binding protein according to the presentdisclosure. Described herein in one embodiment is an antibody comprisingthe single chain variable fragment CD3 binding protein according to thepresent disclosure, wherein said antibody is a scFv antibody. Anotherembodiment describes a multispecific binding protein or antibodycomprising the single chain variable fragment CD3 binding proteinaccording to the present disclosure and a serum albumin binding domain.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates phage titration on biotin-CD3ε and biotin-HSA.

FIG. 2 illustrates the amino acid sequence of the human CD3 bindingprotein (SEQ ID NO: 22). HC CDR1 is indicated in the first shadedsequence (GFTFNKYAMN (SEQ ID NO: 23)), HC CDR2 is indicated in thesecond shaded sequence (RIRSKYNNYATYYADSVK (SEQ ID NO: 24)), HC CDR3 isindicated in the third shaded sequence (HGNFGNSYISYWAY (SEQ ID NO: 25)),LC CDR1 is indicated in the fourth shaded sequence (GSSTGAVTSGNYPN (SEQID NO: 26)), LC CDR2 is indicated in the fifth shaded sequence (GTKFLAP(SEQ ID NO: 27)), and CDR3 is indicated in the sixth shaded sequence(VLWYSNRWV (SEQ ID NO: 28)).

FIG. 3 illustrates the profiles of the sixteen clones selected for moreprecise K_(d) determinations

FIG. 4 illustrates the temperature of hydrophobic exposure (T_(h)° C.)for several anti-huCD3ε scFv variants.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby Certain definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a”, “an” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including”, “includes”,“having”, “has”, “with”, or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the given value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” should be assumed to mean an acceptable error range for theparticular value.

The terms “individual,” “patient,” or “subject” are usedinterchangeably. None of the terms require or are limited to situationcharacterized by the supervision (e.g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly, or a hospice worker).

As used herein, “elimination half-time” is used in its ordinary sense,as is described in Goodman and Gillman's The Pharmaceutical Basis ofTherapeutics 21-25 (Alfred Goodman Gilman, Louis S. Goodman, and AlfredGilman, eds., 6th ed. 1980). Briefly, the term is meant to encompass aquantitative measure of the time course of drug elimination. Theelimination of most drugs is exponential (i.e., follows first-orderkinetics), since drug concentrations usually do not approach thoserequired for saturation of the elimination process. The rate of anexponential process may be expressed by its rate constant, k, whichexpresses the fractional change per unit of time, or by its half-time,t_(1/2) the time required for 50% completion of the process. The unitsof these two constants are time⁻¹ and time, respectively. A first-orderrate constant and the half-time of the reaction are simply related(k×t_(1/2)=0.693) and may be interchanged accordingly. Since first-orderelimination kinetics dictates that a constant fraction of drug is lostper unit time, a plot of the log of drug concentration versus time islinear at all times following the initial distribution phase (i.e. afterdrug absorption and distribution are complete). The half-time for drugelimination can be accurately determined from such a graph.

As used herein, the term “Percent (%) amino acid sequence identity” withrespect to a sequence is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the specific sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.

The term “Framework” or “FR” residues (or regions) refer to variabledomain residues other than the CDR or hypervariable region residues asherein defined. A “human consensus framework” is a framework whichrepresents the most commonly occurring amino acid residue in a selectionof human immunoglobulin VL or VH framework sequences.

As used herein, “Variable region” or “variable domain” refers to thefact that certain portions of the variable domains differ extensively insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its particular antigen. However, thevariability is not evenly distributed throughout the variable domains ofantibodies. It is concentrated in three segments calledcomplementarity-determining regions (CDRs) or hypervariable regions bothin the light-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework (FR).The variable domains of native heavy and light chains each comprise fourFR regions, largely adopting a β-sheet configuration, connected by threeCDRs, which form loops connecting, and in some cases forming part of,the βsheet structure. The CDRs in each chain are held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen-binding site of antibodies(see Kabat et al., Sequences of Proteins of Immunological Interest,Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Theconstant domains are not involved directly in binding an antibody to anantigen, but exhibit various effector functions, such as participationof the antibody in antibody-dependent cellular toxicity. “Variabledomain residue numbering as in Kabat” or “amino acid position numberingas in Kabat,” and variations thereof, refers to the numbering systemused for heavy chain variable domains or light chain variable domains ofthe compilation of antibodies in Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991). Using this numbering system,the actual linear amino acid sequence may contain fewer or additionalamino acids corresponding to a shortening of, or insertion into, a FR orCDR of the variable domain. For example, a heavy chain variable domainmay include a single amino acid insert (residue 52a according to Kabat)after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b,and 82c, etc according to Kabat) after heavy chain FR residue 82. TheKabat numbering of residues may be determined for a given antibody byalignment at regions of homology of the sequence of the antibody with a“standard” Kabat numbered sequence. It is not intended that CDRs of thepresent disclosure necessarily correspond to the Kabat numberingconvention.

As used herein, the term “binding affinity” refers to the affinity ofthe proteins described in the disclosure to their binding targets, andis expressed numerically using “Kd” values. If two or more proteins areindicated to have comparable binding affinities towards their bindingtargets, then the Kd values for binding of the respective proteinstowards their binding targets, are within +2-fold of each other. If twoor more proteins are indicated to have comparable binding affinitiestowards single binding target, then the Kd values for binding of therespective proteins towards said single binding target, are within+2-fold of each other. If a protein is indicated to bind two or moretargets with comparable binding affinities, then the Kd values forbinding of said protein to the two or more targets are within +2-fold ofeach other. In general, a higher Kd value corresponds to a weakerbinding. In some embodiments, the “Kd” is measured by a radiolabeledantigen binding assay (RIA) or surface plasmon resonance assays using aBIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.). Incertain embodiments, an “on-rate” or “rate of association” or“association rate” or “kon” and an “off-rate” or “rate of dissociation”or “dissociation rate” or “koff” are also determined with the surfaceplasmon resonance technique using a BIAcore™-2000 or a BIAcore™-3000(BIAcore, Inc., Piscataway, N.J.). In additional embodiments, the “Kd”,“kon”, and “koff” are measured using the Octet® Systems (Pall LifeSciences).

Described herein are single chain variable fragment CD3 bindingproteins, pharmaceutical compositions as well as nucleic acids,recombinant expression vectors, and host cells for making such singlechain variable fragment CD3 binding proteins. Also provided are methodsof using the disclosed single chain variable fragment CD3 bindingproteins in the prevention, and/or treatment of diseases, conditions anddisorders. In some embodiments, the single chain variable fragment CD3binding proteins are capable of specifically binding to a CD 3 domain,as well as a target antigen and a half-life extension domain, such as asingle domain binding antibody to human serum albumin (HSA).

CD3 Binding Domain

The specificity of the response of T cells is mediated by therecognition of antigen (displayed in context of a majorhistocompatibility complex, MHC) by the T cell receptor complex. As partof the T cell receptor complex, CD3 is a protein complex that includes aCD3γ (gamma) chain, a CD3δ (delta) chain, and two CD3ε (epsilon) chainswhich are present on the cell surface. CD3 associates with the a (alpha)and β (beta) chains of the T cell receptor (TCR) as well as and CD3 ζ(zeta) altogether to comprise the T cell receptor complex. Clustering ofCD3 on T cells, such as by immobilized anti-CD3 antibodies leads to Tcell activation similar to the engagement of the T cell receptor butindependent of its clone-typical specificity.

In one aspect, the single chain variable fragment CD3 binding proteinsdescribed herein comprise a domain which specifically binds to CD3. Inone aspect, the single chain variable fragment CD3 binding proteinsdescribed herein comprise a domain which specifically binds to humanCD3. In one aspect, the single chain variable fragment CD3 bindingproteins described herein comprise a domain which specifically binds tocynomolgus CD3. In one aspect, the single chain variable fragment CD3binding proteins described herein comprise a domain which binds to humanCD3 and cynomolgus CD3. In some embodiments, the single chain variablefragment CD3 binding proteins described herein comprise a domain whichspecifically binds to CD3γ. In some embodiments, the single chainvariable fragment CD3 binding proteins described herein comprise adomain which specifically binds to CD3δ. In some embodiments, the singlechain variable fragment CD3 binding proteins described herein comprise adomain which specifically binds to CD3ε.

In another aspect is provided a multispecific binding protein comprisinga single chain variable fragment CD3 binding protein according to thepresent disclosure. In some embodiments, the multispecific proteincomprising a single chain variable fragment CD3 binding proteinaccording to the present disclosure specifically binds to the T cellreceptor (TCR). In certain instances, the multispecific proteincomprising a single chain variable fragment CD3 binding proteinaccording to the present disclosure binds the α chain of the TCR. Incertain instances, multispecific protein comprising a single chainvariable fragment CD3 binding protein according to the presentdisclosure binds the β chain of the TCR.

In certain embodiments, the CD3 binding domain of the single chainvariable fragment CD3 binding proteins described herein exhibit not onlypotent CD3 binding affinities with human CD3, but show also excellentcrossreactivity with the respective cynomolgus monkey CD3 proteins. Insome instances, the CD3 binding domain of the single chain variablefragment CD3 binding proteins are cross-reactive with CD3 fromcynomolgus monkey. In certain instances, the Kd for binding human CD3(hKd) is about the same as the Kd for binding cynomolgus CD3 (cKd). Incertain instances, the ratio between hKd and cKd (hKd:cKd) is betweenabout 20:1 to about 1:2.

In some embodiments, the CD3 binding domain of the single chain variablefragment CD3 binding protein can be any domain that binds to CD3including but not limited to domains from a monoclonal antibody, apolyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some instances, it is beneficial for the CD3binding domain to be derived from the same species in which the singlechain variable fragment CD3 binding protein will ultimately be used. Forexample, for use in humans, it may be beneficial for the CD3 bindingdomain of the single chain variable fragment CD3 binding protein tocomprise human or humanized residues from the antigen binding domain ofan antibody or antibody fragment.

Thus, in one aspect, the antigen-binding domain comprises a humanized orhuman antibody or an antibody fragment, or a murine antibody or antibodyfragment. In one embodiment, the humanized or human anti-CD3 bindingdomain comprises one or more (e.g., all three) light chain complementarydetermining region 1 (LC CDR1), light chain complementary determiningregion 2 (LC CDR2), and light chain complementary determining region 3(LC CDR3) of a humanized or human anti-CD3 binding domain describedherein, and/or one or more (e.g., all three) heavy chain complementarydetermining region 1 (HC CDR1), heavy chain complementary determiningregion 2 (CDR2), and heavy chain complementary determining region 3(CDR3) of a humanized or human anti-CD3 binding domain described herein,e.g., a humanized or human anti-CD3 binding domain comprising one ormore, e.g., all three, LC CDRs and one or more, e.g., all three, HCCDRs.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human light chain variable region specific toCD3 where the light chain variable region specific to CD3 compriseshuman or non-human light chain CDRs in a human light chain frameworkregion. In certain instances, the light chain framework region is a λ(lambda) light chain framework. In other instances, the light chainframework region is a κ (kappa) light chain framework.

In some embodiments, the humanized or human anti-CD3 binding domaincomprises a humanized or human heavy chain variable region specific toCD3 where the heavy chain variable region specific to CD3 compriseshuman or non-human heavy chain CDRs in a human heavy chain frameworkregion.

In certain instances, the complementary determining regions of the heavychain and/or the light chain are derived from known anti-CD3 antibodies,such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4),teplizumab (MGA031), visilizumab (Nuvion), SP34, TR-66 or X35-3, VIT3,BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2,TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6,T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1 and WT-31.

In one embodiment, the anti-CD3 binding domain is a single chainvariable fragment (scFv) comprising a light chain and a heavy chain ofan amino acid sequence provided herein. As used herein, “single chainvariable fragment” or “scFv” refers to an antibody fragment comprising avariable region of a light chain and at least one antibody fragmentcomprising a variable region of a heavy chain, wherein the light andheavy chain variable regions are contiguously linked via a shortflexible polypeptide linker, and capable of being expressed as a singlepolypeptide chain, and wherein the scFv retains the specificity of theintact antibody from which it is derived. In an embodiment, the anti-CD3binding domain comprises: a light chain variable region comprising anamino acid sequence having at least one, two or three modifications(e.g., substitutions) but not more than 30, 20 or 10 modifications(e.g., substitutions) of an amino acid sequence of a light chainvariable region provided herein, or a sequence with 95-99% identity withan amino acid sequence provided herein; and/or a heavy chain variableregion comprising an amino acid sequence having at least one, two orthree modifications (e.g., substitutions) but not more than 30, 20 or 10modifications (e.g., substitutions) of an amino acid sequence of a heavychain variable region provided herein, or a sequence with 95-99%identity to an amino acid sequence provided herein. In one embodiment,the humanized or human anti-CD3 binding domain is a scFv, and a lightchain variable region comprising an amino acid sequence describedherein, is attached to a heavy chain variable region comprising an aminoacid sequence described herein, via a scFv linker. The light chainvariable region and heavy chain variable region of a scFv can be, e.g.,in any of the following orientations: light chain variable region-scFvlinker-heavy chain variable region or heavy chain variable region-scFvlinker-light chain variable region.

In some instances, scFvs which bind to CD3 are prepared according toknown methods. For example, scFv molecules can be produced by linking VHand VL regions together using flexible polypeptide linkers. The scFvmolecules comprise a scFv linker (e.g., a Ser-Gly linker) with anoptimized length and/or amino acid composition. Accordingly, in someembodiments, the length of the scFv linker is such that the VH or VLdomain can associate intermolecularly with the other variable domain toform the CD3 binding site. In certain embodiments, such scFv linkers are“short”, i.e. consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12amino acid residues. Thus, in certain instances, the scFv linkersconsist of about 12 or less amino acid residues. In the case of 0 aminoacid residues, the scFv linker is a peptide bond. In some embodiments,these scFv linkers consist of about 3 to about 15, for example 8, 10 or15 contiguous amino acid residues. Regarding the amino acid compositionof the scFv linkers, peptides are selected that confer flexibility, donot interfere with the variable domains as well as allow inter-chainfolding to bring the two variable domains together to form a functionalCD3 binding site. For example, scFv linkers comprising glycine andserine residues generally provide protease resistance. In someembodiments, linkers in a scFv comprise glycine and serine residues. Theamino acid sequence of the scFv linkers can be optimized, for example,by phage-display methods to improve the CD3 binding and production yieldof the scFv. Examples of peptide scFv linkers suitable for linking avariable light chain domain and a variable heavy chain domain in a scFvinclude but are not limited to (GS)_(n)(SEQ ID NO: 96), (GGS)_(n)(SEQ IDNO: 97), (GGGS)_(n)(SEQ ID NO: 98), (GGSG)_(n)(SEQ ID NO: 99),(GGSGG)_(n) (SEQ ID NO: 100), (GGGGS)_(n)(SEQ ID NO: 101),(GGGGG)_(n)(SEQ ID NO: 102), or (GGG)_(n)(SEQ ID NO: 103), wherein n is1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the scFv linker canbe (GGGGS)₄ (SEQ ID NO: 104) or (GGGGS)₃ (SEQ ID NO: 1). Variation inthe linker length may retain or enhance activity, giving rise tosuperior efficacy in activity studies.

In some embodiments, CD3 binding domain of a single chain variablefragment CD3 binding protein has an affinity to CD3 on CD3 expressingcells with a K_(d) of 1000 nM or less, 500 nM or less, 200 nM or less,100 nM or less, 80 nM or less, 50 nM or less, 20 nM or less, 10 nM orless, 5 nM or less, 1 nM or less, or 0.5 nM or less. In someembodiments, the CD3 binding domain of a single chain variable fragmentCD3 binding protein has an affinity to CD3ε, γ, or δ with a K_(d) of1000 nM or less, 500 nM or less, 200 nM or less, 100 nM or less, 80 nMor less, 50 nM or less, 20 nM or less, 10 nM or less, 5 nM or less, 1 nMor less, or 0.5 nM or less. In further embodiments, CD3 binding domainof a single chain variable fragment CD3 binding protein has low affinityto CD3, i.e., about 100 nM or greater.

In certain embodiments, the single chain variable fragment CD3 bindingproteins described herein bind to human CD3 with a human Kd (hKd) and tocynomolgus CD3 with a cyno Kd (cKd). In some embodiments, hKd and cKdare between about between about 1 nM to about 2 nM, about 3 nM to about5 nM, about 6 nM to about 10 nM, about 11 nM to about 20 nM, about 25 nMto about 40 nM, about 40 nM to about 60 nM, about 70 nM to about 90 nM,about 100 nM to about 120 nM, about 125 nM to about 140 nM, about 145 nMto about 160 nM, about 170 nM and to about 200 nM, about 210 nM to about250 nM, about 260 nM to about 300 nM.

In some embodiments, the hKd and cKd of the single chain variablefragment CD3 binding proteins is about the same as the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 1.1 fold to about 1.5 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 1.5 fold to about 2 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 2.5 fold to about 3 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 3 fold to about 5 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 6 fold to about 15 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 15 fold to about 20 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 20 fold to about 50 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 55 fold to about 70 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 75 fold to about 100 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22. Insome embodiments, the hKd and cKd of the single chain variable fragmentCD3 binding proteins is about 120 fold to about 200 fold the Kd of a CD3binding protein having the sequence as set forth is SEQ ID NO. 22.

In some embodiments, the ratio between the hKd and cKd (hKd:cKd) rangesfrom about 20:1 to about 1:2. The affinity to bind to CD3 can bedetermined, for example, by the ability of the single chain variablefragment CD3 binding protein itself or its CD3 binding domain to bind toCD3 coated on an assay plate; displayed on a microbial cell surface; insolution; etc. The binding activity of the single chain variablefragment CD3 binding protein itself or its CD3 binding domain of thepresent disclosure to CD3 can be assayed by immobilizing the ligand(e.g., CD3) or the single chain variable fragment CD3 binding proteinitself or its CD3 binding domain, to a bead, substrate, cell, etc.Agents can be added in an appropriate buffer and the binding partnersincubated for a period of time at a given temperature. After washes toremove unbound material, the bound protein can be released with, forexample, SDS, buffers with a high or low pH, and the like and analyzed,for example, by Surface Plasmon Resonance (SPR).

In some embodiments, the single chain variable fragment CD3 bindingprotein has an amino acid sequence selected from SEQ ID NO. 8, SEQ IDNO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18,SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 94), and SEQ IDNO. 95.

In some embodiments, the single chain variable fragment CD3 bindingprotein has an amino acid sequence set forth as SEQ ID NO. 8, whereinthe hKd is about 3.8 nM, and wherein the cKd is about 3.5 nM. In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 9, wherein the hKd isabout 4.1 nM, and wherein the cKd is about 3.4 nM. In some embodiments,the single chain variable fragment CD3 binding protein has an amino acidsequence set forth as SEQ ID NO. 10, wherein the hKd is about 4.3 nM,and wherein the cKd is about 4.2 nM. In some embodiments, the singlechain variable fragment CD3 binding protein has an amino acid sequenceset forth as SEQ ID NO. 11, wherein the hKd is about 4.7 nM, and whereinthe cKd is about 4.9 nM. In some embodiments, the single chain variablefragment CD3 binding protein has an amino acid sequence set forth as SEQID NO. 12, wherein the hKd is about 6.4 nM, and wherein the cKd is about6.6 nM. In some embodiments, the single chain variable fragment CD3binding protein has an amino acid sequence set forth as SEQ ID NO. 13,wherein the hKd is about 8 nM, and wherein the cKd is about 6.6 nM. Insome embodiments, the single chain variable fragment CD3 binding proteinhas an amino acid sequence set forth as SEQ ID NO. 14, wherein the hKdis about 20 nM, and wherein the cKd is about 17 nM. In some embodiments,the single chain variable fragment CD3 binding protein has an amino acidsequence set forth as SEQ ID NO. 15, wherein the hKd is about 37 nM, andwherein the cKd is about 30 nM. In some embodiments, the single chainvariable fragment CD3 binding protein has an amino acid sequence setforth as SEQ ID NO. 16, wherein the hKd is about 14 nM, and wherein thecKd is about 13 nM. In some embodiments, the single chain variablefragment CD3 binding protein has an amino acid sequence set forth as SEQID NO. 17, wherein the hKd is about 50 nM, and wherein the cKd is about47 nM. In some embodiments, the single chain variable fragment CD3binding protein has an amino acid sequence set forth as SEQ ID NO. 18,wherein the hKd is about 16 nM, and wherein the cKd is about 16 nM. Insome embodiments, the single chain variable fragment CD3 binding proteinhas an amino acid sequence set forth as SEQ ID NO. 19, wherein the hKdis about 46 nM, and wherein the cKd is about 43 nM. In some embodiments,the single chain variable fragment CD3 binding protein has an amino acidsequence set forth as SEQ ID NO. 20, wherein the hKd is about 18 nM, andwherein the cKd is about 17 nM. In some embodiments, the single chainvariable fragment CD3 binding protein has an amino acid sequence setforth as SEQ ID NO. 21, wherein the hKd is about 133 nM, and wherein thecKd is about 134 nM. In some embodiments, the single chain variablefragment CD3 binding protein has an amino acid sequence set forth as SEQID NO. 94, wherein the hKd is about 117 nM, and wherein the cKd is about115 nM. In some embodiments, the single chain variable fragment CD3binding protein has an amino acid sequence set forth as SEQ ID NO. 95,wherein the hKd is about 109 nM, and wherein the cKd is about 103 nM.

In some embodiments, the single chain variable fragment CD3 bindingprotein has an amino acid sequence set forth as SEQ ID NO. 8, whereinthe hKd and cKd are about the same as the Kd towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteinhas an amino acid sequence set forth as SEQ ID NO. 9, wherein the hKdand cKd are about the same as the Kd towards CD3 of a protein which hasthe sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 10, wherein the hKd andcKd are about the same as the Kd towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 11, wherein the hKd andcKd are about the same as the Kd towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 12, wherein the hKd andcKd are about the same as the Kd towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 13, wherein the hKd andcKd are about the same as the Kd towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 14, wherein the hKd andcKd are about 3-fold to about 5-fold higher than the Kd towards CD3 of aprotein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein has an amino acid sequence set forth as SEQ ID NO. 15, whereinthe hKd and cKd are about 3-fold to about 5-fold higher than the Kdtowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein has an amino acid sequence set forth as SEQID NO. 16, wherein the hKd and cKd are about 3-fold to about 5-foldhigher than the Kd towards CD3 of a protein which has the sequence asset forth in wt anti-CD3 (SEQ ID NO. 22). In some embodiments, thesingle chain variable fragment CD3 binding protein has an amino acidsequence set forth as SEQ ID NO. 17, wherein the hKd and cKd are about6-fold to about 15-fold higher than the Kd towards CD3 of a proteinwhich has the sequence as set forth in wt anti-CD3 (SEQ ID NO. 22). Insome embodiments, the single chain variable fragment CD3 binding proteinhas an amino acid sequence set forth as SEQ ID NO. 18, wherein the hKdand cKd are about 3-fold to about 5-fold higher than the Kd towards CD3of a protein which has the sequence as set forth in wt anti-CD3 (SEQ IDNO. 22). In some embodiments, the single chain variable fragment CD3binding protein has an amino acid sequence set forth as SEQ ID NO. 19(2A4), wherein the hKd and cKd are about 6-fold to about 15-fold higherthan the Kd towards CD3 of a protein which has the sequence as set forthin wt anti-CD3 (SEQ ID NO. 22). In some embodiments, the single chainvariable fragment CD3 binding protein has an amino acid sequence setforth as SEQ ID NO. 20, wherein the hKd and cKd are about 3-fold toabout 5-fold higher than the Kd towards CD3 of a protein which has thesequence as set forth in wt anti-CD3 (SEQ ID NO. 22). In someembodiments, the single chain variable fragment CD3 binding protein hasan amino acid sequence set forth as SEQ ID NO. 21, wherein the hKd andcKd are about 20-fold to about 50-fold higher than the Kd towards CD3 ofa protein which has the sequence as set forth in wt anti-CD3 (SEQ ID NO.22). In some embodiments, the single chain variable fragment CD3 bindingprotein has an amino acid sequence set forth as SEQ ID NO. 94, whereinthe hKd and cKd are about 20-fold to about 50-fold higher than the Kdtowards CD3 of a protein which has the sequence as set forth in wtanti-CD3 (SEQ ID NO. 22). In some embodiments, the single chain variablefragment CD3 binding protein has an amino acid sequence set forth as SEQID NO. 95, wherein the hKd and cKd are about 20-fold to about 50-foldhigher than the Kd towards CD3 of a protein which has the sequence asset forth in wt anti-CD3 (SEQ ID NO. 22).

Half-Life Extension Domain

Human serum albumin (HSA) (molecular mass ˜67 kDa) is the most abundantprotein in plasma, present at about 50 mg/ml (600 μM), and has ahalf-life of around 20 days in humans. HSA serves to maintain plasma pH,contributes to colloidal blood pressure, functions as carrier of manymetabolites and fatty acids, and serves as a major drug transportprotein in plasma.

Noncovalent association with albumin extends the elimination half-timeof short lived proteins. For example, a recombinant fusion of an albuminbinding domain to a Fab fragment resulted in a decrease in in vivoclearance of 25- and 58-fold and a half-life extension of 26- and37-fold when administered intravenously to mice and rabbits respectivelyas compared to the administration of the Fab fragment alone. In anotherexample, when insulin is acylated with fatty acids to promoteassociation with albumin, a protracted effect was observed when injectedsubcutaneously in rabbits or pigs. Together, these studies demonstrate alinkage between albumin binding and prolonged action.

In one aspect is provided a multispecific binding protein comprising asingle chain variable fragment CD3 binding protein according to thepresent disclosure and further comprising a half-life extension domain,for example a domain which specifically binds to serum albumin. In someembodiments, the serum albumin binding domain of a single chain variablefragment CD3 binding protein can be any domain that binds to serumalbumin including but not limited to domains from a monoclonal antibody,a polyclonal antibody, a recombinant antibody, a human antibody, ahumanized antibody. In some embodiments, the serum albumin bindingdomain is a single chain variable fragments (scFv), single-domainantibody such as a heavy chain variable domain (VH), a light chainvariable domain (VL) and a variable domain (VHH) of camelid derivedsdAb, or antigen binding fragments of the HSA binding antibodies, suchas Fab, Fab′, F(ab)2, and Fv fragments, fragments comprised of one ormore CDRs, single-chain antibodies (e.g., single chain Fv fragments(scFv)), disulfide stabilized (dsFv) Fv fragments, heteroconjugateantibodies (e.g., bispecific antibodies), pFv fragments, heavy chainmonomers or dimers, light chain monomers or dimers, and dimersconsisting of one heavy chain and one light chain, peptide, ligand orsmall molecule entity specific for serum albumin. In certainembodiments, the HSA binding domain is a single-domain antibody. Inother embodiments, the serum albumin binding domain is a peptide. Infurther embodiments, the serum albumin binding domain is a smallmolecule. It is contemplated that the serum albumin binding domain ofthe multispecific binding protein comprising a single chain variablefragment CD3 binding protein is fairly small and no more than 25 kD, nomore than 20 kD, no more than 15 kD, or no more than 10 kD in someembodiments. In certain instances, the serum albumin binding is 5 kD orless if it is a peptide or small molecule entity.

The half-life extension domain of a multispecific binding proteincomprising a single chain variable fragment CD3 binding protein providesfor altered pharmacodynamics and pharmacokinetics of the single chainvariable fragment CD3 binding protein itself. As above, the half-lifeextension domain extends the elimination half-time. The half-lifeextension domain also alters pharmacodynamic properties includingalteration of tissue distribution, penetration, and diffusion of thesingle chain variable fragment CD3 binding protein. In some embodiments,the half-life extension domain provides for improved tissue (includingtumor) targeting, tissue distribution, tissue penetration, diffusionwithin the tissue, and enhanced efficacy as compared with a proteinwithout a half-life extension domain. In one embodiment, therapeuticmethods effectively and efficiently utilize a reduced amount of themultispecific binding protein comprising a single chain variablefragment CD3 binding protein, resulting in reduced side effects, such asreduced non-tumor cell cytotoxicity.

Further, the binding affinity of the half-life extension domain can beselected so as to target a specific elimination half-time in aparticular multispecific binding protein comprising a single chainvariable fragment CD3 binding protein. Thus, in some embodiments, thehalf-life extension domain has a high binding affinity. In otherembodiments, the half-life extension domain has a medium bindingaffinity. In yet other embodiments, the half-life extension domain has alow or marginal binding affinity. Exemplary binding affinities includeK_(d) of 10 nM or less (high), between 10 nM and 100 nM (medium), andgreater than 100 nM (low). As above, binding affinities to serum albuminare determined by known methods such as Surface Plasmon Resonance (SPR).

Target Antigen Binding Domain

In addition to the described CD3 and half-life extension domains, themultispecific binding proteins described herein in certain embodimentsalso comprise a domain that binds to a target antigen. A target antigenis involved in and/or associated with a disease, disorder or condition.In particular, a target antigen associated with a proliferative disease,a tumorous disease, an inflammatory disease, an immunological disorder,an autoimmune disease, an infectious disease, a viral disease, anallergic reaction, a parasitic reaction, a graft-versus-host disease ora host-versus-graft disease. In some embodiments, a target antigen is atumor antigen expressed on a tumor cell. Alternatively in someembodiments, a target antigen is associated with a pathogen such as avirus or bacterium.

In some embodiments, a target antigen is a cell surface molecule such asa protein, lipid or polysaccharide. In some embodiments, a targetantigen is a on a tumor cell, virally infected cell, bacteriallyinfected cell, damaged red blood cell, arterial plaque cell, or fibrotictissue cell.

The design of the multispecific binding protein comprising a singlechain variable fragment CD3 binding protein described herein allows thebinding domain to a target antigen to be flexible in that the bindingdomain to a target antigen can be any type of binding domain, includingbut not limited to, domains from a monoclonal antibody, a polyclonalantibody, a recombinant antibody, a human antibody, a humanizedantibody. In some embodiments, the binding domain to a target antigen isa single chain variable fragments (scFv), single-domain antibody such asa heavy chain variable domain (VH), a light chain variable domain (VL)and a variable domain (VHH) of camelid derived sdAb. In otherembodiments, the binding domain to a target antigen is a non-Ig bindingdomain, i.e., antibody mimetic, such as anticalins, affilins, affibodymolecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers,kunitz domain peptides, and monobodies. In further embodiments, thebinding domain to a target antigen is a ligand or peptide that binds toor associates with a target antigen. In yet further embodiments, thebinding domain to a target antigen is a knottin. In yet furtherembodiments, the binding domain to a target antigen is a small molecularentity.

Single Chain Variable Fragment CD3 Binding Protein Modifications

The single chain variable fragment CD3 binding proteins described hereinencompass derivatives or analogs in which (i) an amino acid issubstituted with an amino acid residue that is not one encoded by thegenetic code, (ii) the mature polypeptide is fused with another compoundsuch as polyethylene glycol, or (iii) additional amino acids are fusedto the protein, such as a leader or secretory sequence or a sequence toblock an immunogenic domain and/or for purification of the protein.

Typical modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphatidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Modifications are made anywhere in single chain variable fragment CD3binding proteins described herein, including the peptide backbone, theamino acid side-chains, and the amino or carboxyl termini. Certaincommon peptide modifications that are useful for modification of singlechain variable fragment CD3 binding proteins include glycosylation,lipid attachment, sulfation, gamma-carboxylation of glutamic acidresidues, hydroxylation, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, and ADP-ribosylation.

Polynucleotides Encoding Single Chain Variable Fragment CD3 BindingProteins

Also provided, in some embodiments, are polynucleotide moleculesencoding a single chain variable fragment CD3 binding protein or amultispecific binding protein comprising a single chain variablefragment CD3 binding protein according to the present disclosure. Insome embodiments, the polynucleotide molecules are provided as a DNAconstruct. In other embodiments, the polynucleotide molecules areprovided as a messenger RNA transcript.

The polynucleotide molecules are constructed by known methods such as bycombining the genes encoding the three binding domains either separatedby peptide linkers or, in other embodiments, directly linked by apeptide bond, into a single genetic construct operably linked to asuitable promoter, and optionally a suitable transcription terminator,and expressing it in bacteria or other appropriate expression systemsuch as, for example CHO cells. In the embodiments where the targetantigen binding domain is a small molecule, the polynucleotides containgenes encoding the CD3 binding domain and the half-life extensiondomain. In the embodiments where the half-life extension domain is asmall molecule, the polynucleotides contain genes encoding the domainsthat bind to CD3 and the target antigen. Depending on the vector systemand host utilized, any number of suitable transcription and translationelements, including constitutive and inducible promoters, may be used.The promoter is selected such that it drives the expression of thepolynucleotide in the respective host cell.

In some embodiments, the polynucleotide is inserted into a vector,preferably an expression vector, which represents a further embodiment.This recombinant vector can be constructed according to known methods.Vectors of particular interest include plasmids, phagemids, phagederivatives, virii (e.g., retroviruses, adenoviruses, adeno-associatedviruses, herpes viruses, lentiviruses, and the like), and cosmids.

A variety of expression vector/host systems may be utilized to containand express the polynucleotide encoding the polypeptide of the describedsingle chain variable fragment CD3 binding protein. Examples ofexpression vectors for expression in E. coli are pSKK (Le Gall et al., JImmunol Methods. (2004) 285(1): 111-27) or pcDNA5 (Invitrogen) forexpression in mammalian cells, PICHIAPINK™ Yeast Expression Systems(Invitrogen), BACUVANCE™ Baculovirus Expression System (GenScript).

Thus, the single chain variable fragment CD3 binding proteins asdescribed herein, in some embodiments, are produced by introducing avector encoding the protein as described above into a host cell andculturing said host cell under conditions whereby the protein domainsare expressed, may be isolated and, optionally, further purified.

Production of Single Chain Variable Fragment CD3 Binding Proteins

Disclosed herein, in some embodiments, is a process for the productionof a single chain variable fragment CD3 binding protein. In someembodiments, the process comprises culturing a host transformed ortransfected with a vector comprising a nucleic acid sequence encoding asingle chain variable fragment CD3 binding protein under conditionsallowing the expression of the single chain variable fragment CD3binding protein and recovering and purifying the produced protein fromthe culture.

In additional embodiment is provided a process directed to improving oneor more properties, e.g. affinity, stability, heat tolerance,cross-reactivity, etc., of the single chain variable fragment CD3binding protein and/or the multispecific binding proteins comprising asingle chain variable fragment CD3 binding protein as described herein,compared to a reference binding compound. In some embodiments, aplurality of single-substitution libraries is provided eachcorresponding to a different domain, or amino acid segment of the singlechain variable fragment CD3 binding protein or reference bindingcompound such that each member of the single-substitution libraryencodes only a single amino acid change in its corresponding domain, oramino acid segment. (This allows all of the potential substitutions in alarge protein or protein binding site to be probed with a few smalllibraries.) In some embodiments, the plurality of domains forms orcovers a contiguous sequence of amino acids of the single chain variablefragment CD3 binding protein or a reference binding compound. Nucleotidesequences of different single-substitution libraries overlap with thenucleotide sequences of at least one other single-substitution library.In some embodiments, a plurality of single-substitution libraries aredesigned so that every member overlaps every member of eachsingle-substitution library encoding an adjacent domain.

Binding compounds expressed from such single-substitution libraries areseparately selected to obtain a subset of variants in each library whichhas properties at least as good as those of the reference bindingcompound and whose resultant library is reduced in size. (That is, thenumber of nucleic acids encoding the selected set of binding compoundsis smaller than the number of nucleic acids encoding members of theoriginal single-substitution library) Such properties include, but arenot limited to, affinity to a target compound, stability with respect tovarious conditions such as heat, high or low pH, enzymatic degradation,cross-reactivity to other proteins and the like. The selected compoundsfrom each single-substitution library are referred to hereininterchangeably as “pre-candidate compounds,” or “pre-candidateproteins.” Nucleic acid sequences encoding the pre-candidate compoundsfrom the separate single-substitution libraries are then shuffled in aPCR to generate a shuffled library, using PCR-based gene shufflingtechniques.

An exemplary work flow of the screening process is described herein.Libraries of pre-candidate compounds are generated from singlesubstitution libraries and selected for binding to the targetprotein(s), after which the pre-candidate libraries are shuffled toproduce a library of nucleic acids encoding candidate compounds which,in turn, are cloned into a convenient expression vector, such as aphagemid expression system. Phage expressing candidate compounds thenundergo one or more rounds of selection for improvements in desiredproperties, such as binding affinity to a target molecule. Targetmolecules may be adsorbed or otherwise attached to a surface of a wellor other reaction container, or target molecules may be derivatized witha binding moiety, such as biotin, which after incubation with candidatebinding compounds may be captured with a complementary moiety, such asstreptavidin, bound to beads, such as magnetic beads, for washing. Inexemplary selection regimens, the candidate binding compounds undergo aprolonged wash step so that only candidate compounds with very lowdissociation rates from a target molecule are selected. Exemplary washtimes for such embodiments are at least 8 hours; or in otherembodiments, at least 24 hours; or in other embodiments, at least 48hours; or in other embodiments, at least 72 hours. Isolated clones afterselection are amplified and subjected to an additional cycle ofselection or analyzed, for example by sequencing and by makingcomparative measurements of binding affinity, for example, by ELISA,surface plasmon resonance binding, bio-layer interferometry (e.g. Octetsystem, ForteBio, Menlo Park, Calif.) or the like.

In some embodiments, the process is implemented to identify one or moresingle chain variable fragment CD3 binding protein and/or amultispecific binding protein comprising a single chain variablefragment CD3 binding protein with improved thermal stability, improvedcross reactivity to a selected set of binding targets compared to thatof a reference CD3 binding protein, such as a protein having the aminoacid sequence of SEQ ID NO. 22. Single substitution libraries areprepared by varying codons in the VH and VL regions of the reference CD3binding protein, including both codons in framework regions and in CDRs;in another embodiment, the locations where codons are varied comprisethe CDRs of the heavy and light chains of the reference CD3 bindingprotein, or a subset of such CDRs, such as solely CDR1, solely CDR2,solely CDR3, or pairs thereof. In another embodiment, locations wherecodons are varied occur solely in framework regions. In someembodiments, a library comprises single codon changes solely from areference CD3 binding protein solely in framework regions of both VH andVL numbering in the range of from 10 to 250. In another embodiment, thelocations where codons are varied comprise the CDR3s of the heavy andlight chains of the reference CD3 binding protein, or a subset of suchCDR3 s. In another embodiment, the number of locations where codons ofVH and VL encoding regions are varied are in the range of from 10 to250, such that up to 100 locations are in framework region. Afterpreparation of the single substitution library, as outlined above, thefollowing steps are carried out: (a) expressing separately each memberof each single substitution library as a pre-candidate protein; (b)selecting members of each single substitution library which encodepre-candidate proteins which bind to a binding partner that differs fromthe original binding target [e.g. a desired cross-reaction target(s)];(c) shuffling members of the selected libraries in a PCR to produce acombinatorial shuffled library; (d) expressing members of the shuffledlibrary as candidate CD3 binding proteins; and (e) selecting members ofthe shuffled library one or more times for candidate CD3 bindingproteins which bind the original binding partner and (f) furtherselecting the candidate proteins for binding to the desiredcross-reactive target(s) thereby providing a nucleic acid encoded CD3binding protein with increased cross reactivity for the one or moresubstances with respect to the reference CD3 binding protein withoutloss of affinity for the original ligand. In additional embodiments, themethod may be implemented for obtaining a single chain variable fragmentCD3 binding protein with decreased reactivity to a selectedcross-reactive substance(s) or compound(s) or epitope(s) by substitutingstep (f) with the following step: depleting candidate binding compoundsone or more times from the subset of candidate single chain variablefragment CD3 binding protein which bind to the undesired cross-reactivecompound.

Pharmaceutical Compositions

Also provided, in some embodiments, are pharmaceutical compositionscomprising a single chain variable fragment CD3 binding proteindescribed herein, a vector comprising the polynucleotide encoding thepolypeptide of the single chain variable fragment CD3 binding protein ora host cell transformed by this vector and at least one pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”includes, but is not limited to, any carrier that does not interferewith the effectiveness of the biological activity of the ingredients andthat is not toxic to the patient to whom it is administered. Examples ofsuitable pharmaceutical carriers are well known in the art and includephosphate buffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions etc. Suchcarriers can be formulated by conventional methods and can beadministered to the subject at a suitable dose. Preferably, thecompositions are sterile. These compositions may also contain adjuvantssuch as preservative, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents.

In some embodiments of the pharmaceutical compositions, the single chainvariable fragment CD3 binding protein described herein is encapsulatedin nanoparticles. In some embodiments, the nanoparticles are fullerenes,liquid crystals, liposome, quantum dots, superparamagneticnanoparticles, dendrimers, or nanorods. In other embodiments of thepharmaceutical compositions, the single chain variable fragment CD3binding protein is attached to liposomes. In some instances, the singlechain variable fragment CD3 binding protein are conjugated to thesurface of liposomes. In some instances, the single chain variablefragment CD3 binding protein are encapsulated within the shell of aliposome. In some instances, the liposome is a cationic liposome.

The single chain variable fragment CD3 binding proteins described hereinare contemplated for use as a medicament. Administration is effected bydifferent ways, e.g. by intravenous, intraperitoneal, subcutaneous,intramuscular, topical or intradermal administration. In someembodiments, the route of administration depends on the kind of therapyand the kind of compound contained in the pharmaceutical composition.The dosage regimen will be determined by the attending physician andother clinical factors. Dosages for any one patient depends on manyfactors, including the patient's size, body surface area, age, sex, theparticular compound to be administered, time and route ofadministration, the kind of therapy, general health and other drugsbeing administered concurrently. An “effective dose” refers to amountsof the active ingredient that are sufficient to affect the course andthe severity of the disease, leading to the reduction or remission ofsuch pathology and may be determined using known methods.

Methods of Treatment

Also provided herein, in some embodiments, are methods and uses forstimulating the immune system of an individual in need thereofcomprising administration of a single chain variable fragment CD3binding protein described herein. In some instances, the administrationof a single chain variable fragment CD3 binding protein described hereininduces and/or sustains cytotoxicity towards a cell expressing a targetantigen. In some instances, the cell expressing a target antigen is acancer or tumor cell, a virally infected cell, a bacterially infectedcell, an autoreactive T or B cell, damaged red blood cells, arterialplaques, or fibrotic tissue.

Also provided herein are methods and uses for a treatment of a disease,disorder or condition associated with a target antigen comprisingadministering to an individual in need thereof a single chain variablefragment CD3 binding protein described herein. Diseases, disorders orconditions associated with a target antigen include, but are not limitedto, viral infection, bacterial infection, auto-immune disease,transplant rejection, atherosclerosis, or fibrosis. In otherembodiments, the disease, disorder or condition associated with a targetantigen is a proliferative disease, a tumorous disease, an inflammatorydisease, an immunological disorder, an autoimmune disease, an infectiousdisease, a viral disease, an allergic reaction, a parasitic reaction, agraft-versus-host disease or a host-versus-graft disease. In oneembodiment, the disease, disorder or condition associated with a targetantigen is cancer. In one instance, the cancer is a hematologicalcancer. In another instance, the cancer is a solid tumor cancer.

As used herein, in some embodiments, “treatment” or “treating” or“treated” refers to therapeutic treatment wherein the object is to slow(lessen) an undesired physiological condition, disorder or disease, orto obtain beneficial or desired clinical results. For the purposesdescribed herein, beneficial or desired clinical results include, butare not limited to, alleviation of symptoms; diminishment of the extentof the condition, disorder or disease; stabilization (i.e., notworsening) of the state of the condition, disorder or disease; delay inonset or slowing of the progression of the condition, disorder ordisease; amelioration of the condition, disorder or disease state; andremission (whether partial or total), whether detectable orundetectable, or enhancement or improvement of the condition, disorderor disease. Treatment includes eliciting a clinically significantresponse without excessive levels of side effects. Treatment alsoincludes prolonging survival as compared to expected survival if notreceiving treatment. In other embodiments, “treatment” or “treating” or“treated” refers to prophylactic measures, wherein the object is todelay onset of or reduce severity of an undesired physiologicalcondition, disorder or disease, such as, for example is a person who ispredisposed to a disease (e.g., an individual who carries a geneticmarker for a disease such as breast cancer).

In some embodiments of the methods described herein, the single chainvariable fragment CD3 binding proteins are administered in combinationwith an agent for treatment of the particular disease, disorder orcondition. Agents include but are not limited to, therapies involvingantibodies, small molecules (e.g., chemotherapeutics), hormones(steroidal, peptide, and the like), radiotherapies (γ-rays, X-rays,and/or the directed delivery of radioisotopes, microwaves, UV radiationand the like), gene therapies (e.g., antisense, retroviral therapy andthe like) and other immunotherapies. In some embodiments, the singlechain variable fragment CD3 binding proteins are administered incombination with anti-diarrheal agents, anti-emetic agents, analgesics,opioids and/or non-steroidal anti-inflammatory agents. In someembodiments, the single chain variable fragment CD3 binding proteins areadministered before, during, or after surgery.

EXAMPLES Example 1: Identification of Anti-CD3 scFv Variants withVarying Affinities for Human CD3ε Characterization of Parental Anti-CD3εPhage

The parental anti-CD3ε showed good binding to biotin-CD3ε and lowbinding to biotin-HSA (FIG. 1).

Anti-CD3ε scFv Phage Libraries

A single substitution library was provided for the heavy chain CDR1,heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2,and light chain CDR3 domains. The amino acid residues varied in eachdomain are illustrated in the highlighted region in FIG. 2. Residueswere varied one at a time via NNN mutagensis.

Selection of Clones and Determination of Binding Affinity

Single substitution libraries were bound to biotinylated hu-CD3ε,washed, eluted, and counted. Biotinylated cynoCD3 was used as the round1selection target, and washed for 4 hours after combinatorial phagebinding from the two independent libraries (˜2x selection). Biotinylatedhu-CD3 was used as the round 2 selection target, and washed for 3 hoursafter binding of both libraries (<2x selection). PCRed inserts from thesecond round of selection were subcloned into the pcDNA3.4 His6expression vector (“His6” disclosed as SEQ ID NO: 105). 180 clones werepicked and DNA was purified, sequenced, and transfected into Expi293. Apanel of sixteen clones with a range of affinities for human CD3ε wereselected for more precise K_(d) determination (FIG. 3).

Example 2: Cytotoxicity Assay

A bispecific antibody directed to CD20 and CD3, containing an anti-CD3scFv variant identified in Example 1 is evaluated in vitro on itsmediation of T cell dependent cytotoxicity to CD20⁺ target cells.

Fluorescence labeled CD20⁺ REC-1 cells (a Mantle cell lymphoma cellline, ATCC CRL-3004) are incubated with isolated PBMC of random donorsor CB 15 T-cells (standardized T-cell line) as effector cells in thepresence of the CD20-CD3 bispecific antibody containing an anti-CD3 scFvvariant identified in Example 1. After incubation for 4 h at 37° C. in ahumidified incubator, the release of the fluorescent dye from the targetcells into the supernatant is determined in a spectrofluorimeter. Targetcells incubated without the CD20-CD3 bispecific antibody containing ananti-CD3 scFv variant identified in Example 1 and target cells totallylysed by the addition of saponin at the end of the incubation serve asnegative and positive controls, respectively.

Based on the measured remaining living target cells, the percentage ofspecific cell lysis is calculated according to the following formula:[1−(number of living targets_((sample))/number of livingtargets_((spontaneous)))]×100%. Sigmoidal dose response curves and EC₅₀values are calculated by non-linear regression/4-parameter logistic fitusing the GraphPad Software. The lysis values obtained for a givenvariant concentration are used to calculate sigmoidal dose-responsecurves by 4 parameter logistic fit analysis using the Prism software.

Example 3: Thermal Stability of Anti-CD3 scFv Variants with VaryingAffinities for Human CD3ε

The temperature of hydrophobic exposure (T_(h)) of a protein correspondsto the derivative of the inflection point of peak dye fluorescence andis known to correlate with melting temperature (Tm), which is a measureof protein stability. The goal of this study was to assess the T_(h) forseveral anti-human CD3ε scFv variants.

Protein Production

Sequences of anti-human CD3ε scFv binding domains were cloned intopcDNA3.4 (Invitrogen) preceded by a leader sequence and followed by a 6×Histidine tag (SEQ ID NO: 105). Expi293F cells (Life TechnologiesA14527) were maintained in suspension in Optimum Growth Flasks (Thomson)between 0.2 to 8×1e6 cells/mL in Expi 293 media. Purified plasmid DNAwas transfected into Expi293F cells in accordance with Expi293Expression System Kit (Life Technologies, A14635) protocols, andmaintained for 4-6 days post transfection. Conditioned media waspartially purified by affinity and desalting chromatography. Anti-humanCD3ε scFv proteins were concentrated with Amicon Ultra centrifugalfiltration units (EMD Millipore), applied to Superdex 200 size exclusionmedia (GE Healthcare) and resolved in a neutral buffer containingexcipients. Fraction pooling and final purity were assessed by SDS-PAGEand analytical SEC (size exclusion chromatography). The absorbance ofpurified protein solutions were determined at 280 nm using a SpectraMaxM2 (Molecular Devices) and UV-transparent 96-well plates (Corning 3635)and their concentrations were calculated from molar extinctioncoefficients.

Differential Scanning Fluorimetry

Purified anti-human CD3ε scFv proteins were diluted to cocentrationsranging from 0.2 to 0.25 mg/mL together with 5× SYPRO orange dye (LifeTechnologies S6651) in 0.15% DMSO final concentration in a neutralbuffer containing excipients into MicroAmp EnduraPlate opticalmicroplates and adhesive film (Applied Biosystems 4483485 and 4311971).A plate containing diluted protein and dye mixtures was loaded into anABI 7500 Fast real-time PCR instrument (Applied Biosytems) and subjectedto a multi-step thermal gradient from 25° C. to 95° C. The thermalgradient comprised of a two minute hold at each one degree celsius stepwith excitation at 500 nm and emission collected with a ROX filter.T_(h) in degrees celsius is presented, for several purified anti-humanCD3ε scFv protein variants, in FIG. 4.

SEQ ID NO: Description AASequence 1 Linker GGGGSGGGGSGGGGS 2HC CDR1 with valiant GX₁X₂X₃NX₄YX₅X₆N positions 3 HC CDR2 with variantpositions R I R S X₇ X₈ N X₉ Y X₁₀ T X₁₁ Y X₁₂ D X₁₃ V K 4HC CDR3 with variant positions H X₁₄ N F X₁₅ X₁₆ S X₁₇ I S Y W A X₁₈ 5LC CDR1 with variant positionsX₁₉ X₂₀ X₂₁ X₂₂ G X₂₃ V X₂₄ X₂₅ G X₂₆ Y P N 6 LC CDR2 with variantpositions G X₂₇ X₂₈ X₂₉ X₃₀ X₃₁ P 7 LC CDR3 with variant positionsX₃₂ L W Y X₃₃ N X₃₄ W X₃₅ 8 Anti-CD3, clone 2B2EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEA EYYCTLWYSNRWVFGGGTKLTVL 9Anti-CD3, clone 9F2 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKGLEWVARIRSKYNKYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYDNRWVFGGGTKLTVL10 Anti-CD3, clone 5A2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSHISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGYVTSGNYPNWVQQKPGQAPRGLIGGTSFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWIFGGGTKLTVL11 Anti-CD3, clone 6A2 EVQLVESGGGLVQPGGSLKLSCAASGFMFNKYAMNWVRQAPGKGLEWVARIRSKSNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWATWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSFGAVTSGNYPNWVQQKPGQAPRGLIGGTKLLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNSWVFGGGTKLTVL12 Anti-CD3, clone 2D2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYKDSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL13 Anti-CD3, clone 3F2 EVQLVESGGGLVQPGGSLKLSCAASGFTYNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADEVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSKGAVTSGNYPNWVQQKPGQAPRGLIGGTKELAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCTLWYSNRWVFGGGTKLTVL14 Anti-CD3, clone 1A2 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHTNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTYFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL15 Anti-CD3, clone 1C2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADAVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSQISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTDGNYPNWVQQKPGQAPRGLIGGIKFLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVL16 Anti-CD3, clone 2E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAVNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGESTGAVTSGNYPNWVQQKPGQAPRGLIGGTKILAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL17 Anti-CD3, clone 10E4 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYPMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKNEDTAVYYCVRHGNFNNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTKGNYPNWVQQKPGQAPRGLIGGTKMLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCALWYSNRWVFGGGTKLTVL18 Anti-CD3, clone 2H2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADEVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVVSGNYPNWVQQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVL19 Anti-CD3, clone 2A4 EVQLVESGGGLVQPGGSLKLSCAASGNTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGDSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTHGNYPNWVQQKPGQAPRGLIGGTKVLAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYSNRWVFGGGTKLTVL20 Anti-CD3, clone 10B2 EVQLVESGGGLVQPGGSLKLSCAASGFTFNNYAMNWVRQAPGKGLEWVARIRSGYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSYTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFNAPGTPARFSGSLLGGKAALTLSGVQPED EAEYYCVLWYANRWVFGGGTKLTVL21 Anti-CD3, clone 1G4 EVQLVESGGGLVQPGGSLKLSCAASGFEFNKYAMNWVRQAPGKGLEWVARIRSKYNNYETYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSLISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSSGAVTSGNYPNWVQQKPGQAPRGLIGGTKFGAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL22 wt anti-CD3 EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL23 wt anti-CD3 HC CDR1 GFTFNKYAMN 24 wt anti-CD3 HC CDR2RIRSKYNNYATYYADSVK 25 wt anti-CD3 HC CDR3 HGNFGNSYISYWAY 26wt anti-CD3 LC CDR1 GSSTGAVTSGNYPN 27 wt anti-CD3 LC CDR2 GTKFLAP 28wt anti-CD3 LC CDR3 VLWYSNRWV 29 HC CDR1 variant 1 GNTFNKYAMN 30HC CDR1 variant 2 GFEFNKYAMN 31 HC CDR1 variant 3 GFMFNKYAMN 32HC CDR1 variant 4 GFTYNKYAMN 33 HC CDR1 variant 5 GFTFNNYAMN 34HC CDR1 variant 6 GFTFNGYAMN 35 HC CDR1 variant 7 GFTFNTYAMN 36HC CDR1 variant 8 GFTFNEYAMN 37 HC CDR1 variant 9 GFTFNKYPMN 38HC CDR1 variant 10 GFTFNKYAVN 39 HC CDR1 variant 11 GFTFNKYAIN 40HC CDR1 variant 12 GFTFNKYALN 41 HC CDR2 variant 1 RIRSGYNNYATYYADSVK 42HC CDR2 variant 2 RIRSKSNNYATYYADSVK 43 HC CDR2 variant 3RIRSKYNKYATYYADSVK 44 HC CDR2 variant 4 RIRSKYNNYETYYADSVK 45HC CDR2 variant 5 RIRSKYNNYATEYADSVK 46 HC CDR2 variant 6RIRSKYNNYATYYKDSVK 47 HC CDR2 variant 7 RIRSKYNNYATYYADEVK 48HC CDR2 variant 8 RIRSKYNNYATYYADAVK 49 HC CDR2 variant 9RIRSKYNNYATYYADQVK 50 HC CDR2 variant 10 RIRSKYNNYATYYADDVK 51HC CDR3 variant 1 HANFGNSYISYWAY 52 HC CDR3 variant 2 HTNFGNSYISYWAY 53HC CDR3 variant 3 HGNFNNSYISYWAY 54 HC CDR3 variant 4 HGNFGDSYISYWAY 55HC CDR3 variant 5 HGNFGNSHISYWAY 56 HC CDR3 variant 6 HGNFGNSPISYWAY 57HC CDR3 variant 7 HGNFGNSQISYWAY 58 HC CDR3 variant 8 HGNFGNSLISYWAY 59HC CDR3 variant 9 HGNFGNSGISYWAY 60 HC CDR3 variant 10 HGNFGNSYISYWAT 61LC CDR1 variant 1 ASSTGAVTSGNYPN 62 LC CDR1 variant 2 GESTGAVTSGNYPN 63LC CDR1 variant 3 GSYTGAVTSGNYPN 64 LC CDR1 variant 4 GSSFGAVTSGNYPN 65LC CDR1 variant 5 GSSKGAVTSGNYPN 66 LC CDR1 variant 6 GSSSGAVTSGNYPN 67LC CDR1 variant 7 GSSTGYVTSGNYPN 68 LC CDR1 variant 8 GSSTGAVVSGNYPN 69LC CDR1 variant 9 GSSTGAVTDGNYPN 70 LC CDR1 variant 10 GSSTGAVTKGNYPN 71LC CDR1 variant 11 GSSTGAVTHGNYPN 72 LC CDR1 variant 12 GSSTGAVTVGNYPN73 LC CDR1 variant 13 GSSTGAVTSGYYPN 74 LC CDR2 variant 1 GIKFLAP 75LC CDR2 variant 2 GTEFLAP 76 LC CDR2 variant 3 GTYFLAP 77LC CDR2 variant 4 GTSFLAP 78 LC CDR2 variant 5 GTNFLAP 79LC CDR2 variant 6 GTKLLAP 80 LC CDR2 variant 7 GTKELAP 81LC CDR2 variant 8 GTKILAP 82 LC CDR2 variant 9 GTKMLAP 83LC CDR2 variant 10 GTKVLAP 84 LC CDR2 variant 11 GTKFNAP 85LC CDR2 variant 12 GTKFGAP 86 LC CDR2 variant 13 GTKFLVP 87LC CDR3 variant 1 TLWYSNRWV 88 LC CDR3 variant 2 ALWYSNRWV 89LC CDR3 variant 3 VLWYDNRWV 90 LC CDR3 variant 4 VLWYANRWV 91LC CDR3 variant 5 VLWYSNSWV 92 LC CDR3 variant 6 VLWYSNRWI 93LC CDR3 variant 7 VLWYSNRWA 94 Anti-CD3, clone 2G5EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYALNWVRQAPGKGLEWVARIRSKYNNYATEYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSPISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTNFLAPGTPERFSGSLLGGKAALTLSGVQPEDEA EYYCVLWYSNRWAFGGGTKLTVL 95 Anti-CD3, clone 8A5 EVQLVESGGGLVQPGGSLKLSCAASGFTFNEYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADDVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSGISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTVGNYPNWVQQKPGQAPRGLIGGTEFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL96 Exemplary linker sequence (GS)n 97 Exemplary linker sequence (GGS)n98 Exemplary linker sequence (GGGS)n 99 Exemplary linker sequence(GGSG)n 100 Exemplary linker sequence (GGSGG)n 101Exemplary linker sequence (GGGGS)n 102 Exemplary linker sequence(GGGGG)n 103 Exemplary linker sequence (GGG)n 104Exemplary linker sequence (GGGGS)4 105 6X Histidine HHHHHH

1.-30. (canceled)
 31. A method of treating a cancer, the methodcomprising administering to a subject a single chain variable fragmentCD3 binding protein comprising a variable heavy chain region (VH), avariable light chain region (VL), and a linker, wherein the VH comprisescomplementarity determining regions HC CDR1, HC CDR2, and HC CDR3,wherein the VL comprises complementarity determining regions LC CDR1, LCCDR2, and LC CDR3, wherein the protein is at least eighty percentidentical to SEQ ID NO: 22, wherein (a) the amino acid sequence of HCCDR1 is as set forth in SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36,SEQ ID NO. 37, SEQ ID NO.38, SEQ ID NO. 39, or SEQ ID NO. 40; (b) theamino acid sequence of HC CDR2 is as set forth in SEQ ID NO. 41, SEQ IDNO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, or SEQ ID NO. 50; (c) the aminoacid sequence of HC CDR3 is as set forth in SEQ ID NO. 51, SEQ ID NO.52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ IDNO. 57, SEQ ID NO. 58, SEQ ID NO. 59, or SEQ ID NO. 60; (d) the aminoacid sequence of LC CDR1 is as set forth in SEQ ID NO. 61, SEQ ID NO.62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ IDNO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQID NO. 72, or SEQ ID NO: 73; (e) the amino acid sequence of LC CDR2 isas set forth in SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO.77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ IDNO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, or SEQ ID NO. 86;and (f) the amino acid sequence of LC CDR3 is as set forth in SEQ ID NO.87, SEQ ID NO: 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ IDNO. 92, or SEQ ID NO. 93, and wherein said protein has an amino acidsequence that is at least 95% identical to a sequence selected from thegroup consisting of: SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ IDNO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20,SEQ ID NO. 21, SEQ ID NO. 94, and SEQ ID NO.
 95. 32. The method of claim31, wherein HC CDR1 is SEQ ID NO. 39, HC CDR2 is SEQ ID NO. 49, HC CDR3is SEQ ID NO. 51, LC CDR1 is SEQ ID NO. 61, LC CDR2 is SEQ ID NO. 86,and LC CDR3 is SEQ ID NO.
 87. 33. The method of claim 31, wherein HCCDR1 is SEQ ID NO. 30, HC CDR2 is SEQ ID NO. 43, LC CDR1 is SEQ ID NO.64, and LC CDR3 is SEQ ID NO.
 89. 34. The method of claim 31, wherein HCCDR3 is SEQ ID NO. 55, LC CDR1 is SEQ ID NO. 67, LC CDR2 is SEQ ID NO.77, and LC CDR3 is SEQ ID NO.
 92. 35. The method of claim 31, wherein HCCDR1 is SEQ ID NO. 31, HC CDR2 is SEQ ID NO. 42, HC CDR3 is SEQ ID NO.60, LC CDR1 is SEQ ID NO. 64, LC CDR2 is SEQ ID NO. 79, and LC CDR3 isSEQ ID NO.
 91. 36. The method of claim 31, wherein HC CDR1 is SEQ ID NO.35, HC CDR2 is SEQ ID NO. 46, HC CDR3 is SEQ ID NO. 56, LC CDR1 is SEQID NO. 68, and LC CDR3 is SEQ ID NO.
 75. 37. The method of claim 31,wherein HC CDR1 is SEQ ID NO. 29, HC CDR2 is SEQ ID NO. 44, HC CDR3 isSEQ ID NO. 52, LC CDR1 is SEQ ID NO. 73, and LC CDR2 is SEQ ID NO. 76.38. The method of claim 31, wherein HC CDR1 is SEQ ID NO. 33, HC CDR2 isSEQ ID NO. 48, HC CDR3 is SEQ ID NO. 57, LC CDR1 is SEQ ID NO. 69, andLC CDR2 is SEQ ID NO.
 74. 39. The method of claim 31, wherein HC CDR1 isSEQ ID NO. 37, HC CDR3 is SEQ ID NO. 53, LC CDR1 is SEQ ID NO. 70, LCCDR2 is SEQ ID NO. 82, and LC CDR3 is SEQ ID NO.
 88. 40. The method ofclaim 31, wherein HC CDR1 is SEQ ID NO. 34, HC CDR2 is SEQ ID NO. 47, HCCDR3 is SEQ ID NO. 56, LC CDR1 is SEQ ID NO. 68 and LC CDR2 is SEQ IDNO.
 75. 41. The method of claim 31, wherein HC CDR1 is SEQ ID NO. 29, HCCDR3 is SEQ ID NO. 54, LC CDR1 is SEQ ID NO. 71 and LC CDR2 is SEQ IDNO.
 83. 42. The method of claim 31, wherein HC CDR1 is SEQ ID NO. 33, HCCDR2 is SEQ ID NO. 41, LC CDR1 is SEQ ID NO. 63, LC CDR2 is SEQ ID NO.84, and LC CDR1 is SEQ ID NO. 90 or wherein HC CDR1 is SEQ ID NO. 30, HCCDR2 is SEQ ID NO. 44, HC CDR3 is SEQ ID NO. 58, LC CDR1 is SEQ ID NO.66 and LC CDR2 is SEQ ID NO.
 85. 43. The method of claim 31, wherein HCCDR1 is SEQ ID NO. 40, HC CDR2 is SEQ ID NO. 45, HC CDR3 is SEQ ID NO.56, LC CDR2 is SEQ ID NO. 78 and LC CDR3 is SEQ ID NO. 93 or wherein HCCDR1 is SEQ ID NO. 36, HC CDR2 is SEQ ID NO. 50, HC CDR3 is SEQ ID NO.59, LC CDR1 is SEQ ID NO. 72 and LC CDR2 is SEQ ID NO.
 75. 44. Themethod of claim 31, wherein the protein comprises a sequence that is atleast 80% identical to SEQ ID NO.
 22. 45. The method of claim 31,wherein the protein comprises a sequence selected from the groupconsisting of SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11,SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO.16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ IDNO. 21, SEQ ID NO. 94, and SEQ ID NO.
 95. 46. The method of claim 45,wherein the protein comprises the sequence of SEQ ID No.
 8. 47. Themethod of claim 31, wherein the linker has a sequence selected fromthose of SEQ ID NO. 1 and SEQ ID NO. 96-104.
 48. The method of claim 31,wherein the cancer comprises a hematological cancer.
 49. The method ofclaim 31, wherein the cancer comprises a solid tumor cancer.
 50. Amethod of treating a cancer, the method comprising administering asingle chain variable fragment CD3 binding protein comprising a variableheavy chain region (VH) and a variable light chain region (VL), whereinthe VH comprises complementarity determining regions HC CDR1, HC CDR2,and HC CDR3, wherein the VL comprises complementarity determiningregions LC CDR1, LC CDR2, and LC CDR3, and wherein the HC-CDR1, theHC-CDR2, the HC-CDR3, the LC-CDR1, the LC-CDR1, and the LC-CDR3 are notsimultaneously SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO.26, SEQ ID NO. 27, and SEQ ID NO. 28, and wherein the protein is atleast 80% identical to SEQ ID NO.
 22. 51. The method of claim 50,wherein (i) HC CDR1 is SEQ ID NO. 39, HC CDR2 is SEQ ID NO. 49, HC CDR3is SEQ ID NO. 51, LC CDR1 is SEQ ID NO. 61, LC CDR2 is SEQ ID NO. 86,and LC CDR3 is SEQ ID NO. 87; (ii) HC CDR1 is SEQ ID NO. 30, HC CDR2 isSEQ ID NO. 43, HC CDR3 is SEQ ID NO. 25, LC CDR1 is SEQ ID NO. 64, LCCDR2 is SEQ ID NO. 27, and LC CDR3 is SEQ ID NO. 89; (iii) HC CDR1 isSEQ ID NO. 23, HC CDR2 is SEQ ID NO. 24, HC CDR3 is SEQ ID NO. 55, LCCDR1 is SEQ ID NO. 67, LC CDR2 is SEQ ID NO. 77, and LC CDR3 is SEQ IDNO. 92; (iv) HC CDR1 is SEQ ID NO. 31, HC CDR2 is SEQ ID NO. 42, HC CDR3is SEQ ID NO. 60, LC CDR1 is SEQ ID NO. 64, LC CDR2 is SEQ ID NO. 79,and LC CDR3 is SEQ ID NO. 91; (v) HC CDR1 is SEQ ID NO. 35, HC CDR2 isSEQ ID NO. 46, HC CDR3 is SEQ ID NO. 56, LC CDR1 is SEQ ID NO. 68, LCCDR2 is SEQ ID NO. 75, and LC CDR3 is SEQ ID NO. 28; (vi) HC CDR1 is SEQID NO. 32, HC CDR2 is SEQ ID NO. 47, HC CDR3 is SEQ ID NO. 56, LC CDR1is SEQ ID NO. 65, LC CDR2 is SEQ ID NO. 80, and LC CDR3 is SEQ ID NO.87; (vii) HC CDR1 is SEQ ID NO. 29 HC CDR2 is SEQ ID NO. 44, HC CDR3 isSEQ ID NO. 52, LC CDR1 is SEQ ID NO. 73, LC CDR2 is SEQ ID NO. 76, andLC CDR3 is SEQ ID NO. 28; (viii) HC CDR1 is SEQ ID NO. 33, HC CDR2 isSEQ ID NO. 48, HC CDR3 is SEQ ID NO. 57, LC CDR1 is SEQ ID NO. 69, LCCDR2 is SEQ ID NO. 74, and LC CDR3 is SEQ ID NO. 28; (ix) HC CDR1 is SEQID NO. 38, HC CDR2 is SEQ ID NO. 24, HC CDR3 is SEQ ID NO. 25, LC CDR1is SEQ ID NO. 62, LC CDR2 is SEQ ID NO. 81, and LC CDR3 is SEQ ID NO.28; (x) HC CDR1 is SEQ ID NO. 37, HC CDR2 is SEQ ID NO. 24, HC CDR3 isSEQ ID NO. 53, LC CDR1 is SEQ ID NO. 70, LC CDR2 is SEQ ID NO. 82, andLC CDR3 is SEQ ID NO. 88; (xi) HC CDR1 is SEQ ID NO. 34, HC CDR2 is SEQID NO. 47, HC CDR3 is SEQ ID NO. 56, LC CDR1 is SEQ ID NO. 68, LC CDR2is SEQ ID NO. 75, and LC CDR3 is SEQ ID NO. 28; (xii) HC CDR1 is SEQ IDNO. 29, HC CDR2 is SEQ ID NO. 24, HC CDR3 is SEQ ID NO. 54, LC CDR1 isSEQ ID NO. 71, LC CDR2 is SEQ ID NO. 83, and LC CDR3 is SEQ ID NO. 28;(xiii) HC CDR1 is SEQ ID NO. 33, HC CDR2 is SEQ ID NO. %1, HC CDR3 isSEQ ID NO. 25, LC CDR1 is SEQ ID NO. 63, LC CDR2 is SEQ ID NO. 34, andLC CDR3 is SEQ ID NO. 90; (xiv) HC CDR 1 is SEQ ID NO. 30, HC CDR2 isSEQ ID NO. 44, HC CDR3 is SEQ ID NO. 58, LC CDR1 is SEQ ID NO. 66, LCCDR2 is SEQ ID NO. 85, and LC CDR3 is SEQ ID NO. 28; (xv) HC CDR1 is SEQID NO. 94, HC CDR2 is SEQ ID NO. 45, HC CDR3 is SEQ ID NO. 56, LC CDR1is SEQ ID NO. 26, LC CDR2 is SEQ ID NO. 78, and LC CDR3 is SEQ ID NO.93; or (xvi) HC CDR1 is SEQ ID NO. 36, HC CDR2 is SEQ ID NO. 50, HC CDR3is SEQ ID NO. 59, LC CDR1 is SEQ ID NO. 72, LC CDR2 is SEQ ID NO. 75,and LC CDR3 is SEQ ID NO.
 28. 52. The method of claim 50, wherein theprotein comprises a sequence that is at least 80% identical to asequence selected from the group consisting of SEQ ID NO. 8, SEQ ID NO.9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ IDNO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 94, and SEQ ID NO.95.
 53. The method of claim 50, wherein the cancer comprises ahematological cancer.
 54. The method of claim 50, wherein the cancercomprises a solid tumor cancer.
 55. A method of treating a cancercomprising administering a multispecific protein comprising: (a) asingle chain variable fragment CD3 binding protein, (b) a domain thatspecifically binds to a serum albumin, and (c) a target antigen bindingdomain, wherein the CD3 binding protein comprising a variable heavychain region (VH), a variable light chain region (VL), wherein VHcomprises complementarity determining regions HC CDR1, HC CDR2, and HCCDR3, wherein VL comprises complementarity determining regions LC CDR1,LC CDR2, and LC CDR3, wherein (i) HC CDR1 is SEQ ID NO. 39, HC CDR2 isSEQ ID NO. 49, HC CDR3 is SEQ ID NO. 51, LC CDR1 is SEQ ID NO. 61, LCCDR2 is SEQ ID NO. 86, and LC CDR3 is SEQ ID NO. 87, (ii) HC CDR1 is SEQID NO. 30, HC CDR2 is SEQ ID NO. 43, HC CDR3 is SEQ ID NO. 25, LC CDR1is SEQ ID NO. 64, LC CDR2 is SEQ ID NO. 27, and LC CDR3 is SEQ ID NO.89; (iii) HC CDR1 is SEQ ID NO. 23, HC CDR2 is SEQ ID NO. 24, HC CDR3 isSEQ ID NO. 55, LC CDR1 is SEQ ID NO. 67, LC CDR2 is SEQ ID NO. 77, andLC CDR3 is SEQ ID NO. 92; (iv) HC CDR1 is SEQ ID NO. 31, HC CDR2 is SEQID NO. 42, HC CDR3 is SEQ ID NO. 60, LC CDR1 is SEQ ID NO. 64, LC CDR2is SEQ ID NO. 79, and LC CDR3 is SEQ ID NO. 91; (v) HC CDR-1 is SEQ IDNO. 35, HC CDR2 is SEQ ID NO. 46, HC CDR3 is SEQ ID NO. 56, LC CDR1 isSEQ ID NO. 68, LC CDR2 is SEQ ID NO. 75, and LC CDR3 is SEQ ID NO. 28;(vi) HC CDR1 is SEQ ID NO. 32, HC CDR2 is SEQ ID NO. 47, HC CDR3 is SEQID NO. 56, LC CDR1 is SEQ NO. 65, L CDR2 is SEQ ID NO. 80, and LC CDR3is SEQ ID NO. 87; (vii) HC CDR1 is SEQ ID NO. 29, HC CDR2 is SEQ ID NO.44, HC CDR3 is SEQ ID NO. 52, LC CDR1 is SEQ ID NO. 73, LC CDR2 is SEQID NO. 76, and LC CDR3 is SEQ ID NO. 28; (viii) HC CDR1 is SEQ ID NO.33, HC CDR2 is SEQ ID NO. %18, HC CDR3 is SEQ ID NO. 57, LC CDR1 is SEQID NO. 69, LC CDR2 is SEQ ID NO. 74, and LC CDR3 is SEQ ID NO. 28; (ix)HC CDR1 is SEQ ID NO. 38, HC CDR2 is SEQ ID NO, 24, HC CDR3 is SEQ IDNO. 25, LC CDR1 is SEQ ID NO. 62, LC CDR2 is SEQ ID NO. 81, and LC CDR3is SEQ ID NO. 28; (x) HC CDR1 is SEQ ID NO. 37, HC CDR2 is SEQ ID NO.24, HC CDR3 is SEQ ID NO. 53, LC CDR1 is SEQ ID NO. 70 LC CDR2 is SEQ IDNO. 82, and LC CDR3 is SEQ ID NO. 88; (xi) HC CDR1 is SEQ ID NO. 3, HCCDR2 is SEQ ID NO. 47, HC CDR3 is SEQ ID NO. 56, LC CDR1 is SEQ ID NO.68, LC CDR2 is SEQ ID NO. 75, and LC CDR3 is SEQ ID NO. 28; (xii) HCCDR1 is SEQ ID NO. 29 HC CDR2 is SEQ ID NO. 24, HC CDR3 is SEQ ID NO.54, LC CDR1 is SEQ ID NO. 71, LC CDR2 is SEQ ID NO. 33, and LC CDR3 isSEQ ID NO. 28; (xiii) HC CDR1 is SEQ ID NO. 33, HC CDR2 is SEQ ID NO.41, HC CDR3 is SEQ ID NO. 25, LC CDR1 is SEQ ID NO. 63, LC CDR2 is SEQID NO. 84, and LC CDR3 is SEQ ID NO. 90; (xiv) HC CDR1 is SEQ ID NO. 30,HC CDR2 is SEQ ID NO. 44, HC CDR3 is SEQ ID NO, 58, LC CDR1 is SEQ IDNO. 66, LC CDR2 is SEQ ID NO. 85, and LC CDR3 is SEQ ID NO. 28; (xv) HCCDR1 is SEQ ID NO. 94, HC CDR2 is SEQ ID NO. 45, HC CDR3 is SEQ ID NO.56, LC CDR1 is SEQ ID NO. 26, LC CDR2 is SEQ ID NO. 78, and LC CDR3 isSEQ ID NO. 93; or (xvi) HC CDR1 is SEQ ID NO. 36, HC CDR2 is SEQ ID NO.50, HC CDR3 is SEQ ID NO. 59, LC CDR1 is SEQ ID NO. 72, LC CDR2 is SEQID NO. 75, and LC CDR3 is SEQ ID NO. 28.